Silk-hyaluronic acid based tissue fillers and methods of using the same

ABSTRACT

Hyaluronic acid and silk protein fragments based tissue fillers and methods of using the same are provided herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplications Nos. 62/525,131, filed on Jun. 26, 2017, and 62/641,095,filed on Mar. 9, 2018, which are incorporated by reference herein intheir entireties.

BACKGROUND OF THE INVENTION

Silk is a natural polymer produced by a variety of insects and spiders.Silk comprises a filament core protein, silk fibroin, and a glue-likecoating consisting of a non-filamentous protein, sericin. Silk has beenhistorically studied for use in the medical field. Hyaluronic acid(hyaluronan) is a glycosaminoglycan that is distributed throughout thebody and is found in connective and epithelial tissues. Due to itsbiocompatibility and structural benefits, it is a useful component inmedical devices and implantable materials.

Soft tissues of the human body owe their structures in part to anextracellular matrix that includes collagen, elastin, andglycosaminoglycan. Soft tissue defects may occur, which distort, deform,or otherwise alters soft tissue structures. Such structure may berestored through the use of tissue fillers that may be deposited at thedefect site remedy the defect. For example, tissue fillers may be placedat the site of a facial wrinkle to remedy the wrinkle.

However, new tissue fillers are needed in the field that remedy a numberof tissue defects while providing tunable properties, which may allowfor tailoring of the tissue filler to the specific tissue defect.

SUMMARY OF THE INVENTION

In one embodiment, the invention relates to a biocompatible tissuefiller comprising: a glycosaminoglycan selected from the groupconsisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC),starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthangum, chitosan, pectin, agar, carrageenan, and guar gum: and an activeagent selected from the group consisting of an enzyme inhibitor, ananesthetic agent, a medicinal neurotoxin, an antioxidant, ananti-infective agent, an anti-inflammatory agent, an ultraviolet (UV)light blocking agent, a dye, a hormone, an immunosuppressant, and ananti-inflammatory agent; wherein a portion of the glycosaminoglycan iscross-linked by cross-linking moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol; andwherein cross-linking is obtained using a cross-linking agent, across-linking precursor, or an activating agent. In some embodiments,the glycosaminoglycan is hyaluronic acid (HA). In some embodiments, the% w/w amount of cross-linked HA relative to the total amount of HA isabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%,about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100%. In some embodiments, the degree ofcross-linking of the cross-linked HA is between about 1% and about 100%.In some embodiments, the degree of cross-linking of the cross-linked HAis about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%,about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100%. In some embodiments, the degree ofcross-linking of the cross-linked HA is between about 1% and about 15%.In some embodiments, the degree of cross-linking of the cross-linked HAis one or more of about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, and about 15%.

In some embodiments, the cross-linked HA comprises a cross-linkingmoiety comprising a polyethylene glycol (PEG) chain. In someembodiments, the cross-linking agent and/or the cross-linking precursorcomprises an epoxy group. In some embodiments, cross-linking is obtainedusing a cross-linking agent, a cross-linking precursor, or an activatingagent selected from the group consisting of a polyepoxy linker, adiepoxy linker, a polyepoxy-PEG, a diepoxy-PEG, a polyglycidyl-PEG, adiglycidyl-PEG, a poly acrylate PEG, a diacrylate PEG,1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, divinylsulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light,glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE),1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritoltetraglycidyl ether (PETGE), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMIDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, a carbodiimide, and anycombinations thereof. In some embodiments, cross-linking is obtainedusing a polyfunctional epoxy compound selected from the group consistingof 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidylether (EGDGE), 1,6-hexanediol diglycidyl ether, polyethylene glycoldiglycidyl ether, polypropylene glycol diglycidyl ether,polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidylether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether,glycerol polyglycidyl ether, tri-methylolpropane polyglycidyl ether,pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether. Insome embodiments, cross-linking is obtained using a cross-linking agentand/or a cross-linking precursor selected from the group consisting ofpolyethylene glycol diglycidyl ether, diepoxy PEG, PEG diglycidyl ether,polyoxyethylene bis-glycidyl ether, PEGDE, and PEGDGE. In someembodiments, cross-linking is obtained using polyethylene glycoldiglycidyl ether having an average M_(n) of about 500, about 1000, about2000, or about 6000. In some embodiments, cross-linking is obtainedusing polyethylene glycol diglycidyl ether having from 2 to 25 ethyleneglycol groups. In some embodiments, cross-linking is obtained using across-linking agent and/or a cross-linking precursor selected from thegroup consisting of a polyepoxy silk fibroin linker, a diepoxy silkfibroin linker, a polyepoxy silk fibroin fragment linker, a diepoxy silkfibroin fragment linker, a polyglycidyl silk fibroin linker, adiglycidyl silk fibroin linker, a polyglycidyl silk fibroin fragmentlinker, and a diglycidyl silk fibroin fragment linker.

In some embodiments, the invention relates to a tissue filler furthercomprising an organic compound and/or an inorganic compound. In someembodiments, the inorganic compound comprises calcium hydroxyapatite. Insome embodiments, the calcium hydroxyapatite is formulated as particleshaving a diameter between about 1 μm and about 100 μm, between about 1μm and about 10 μm, between about 2 μm and about 12 μm, between about 3μm and about 10 μm, between about 4 μm and about 15 μm, between about 8μm and about 12 μm, between about 5 μm and about 10 μm, between about 6μm and about 12 μm, between about 7 μm and about 20 μm, between about 9μm and about 18 μm, or between about 10 μm and about 25 μm. In someembodiments, the concentration of calcium hydroxyapatite is betweenabout 0.001% and about 5%. In some embodiments, the concentration ofcalcium hydroxyapatite is about 0.001%, about 0.002%, about 0.003%,about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%,about 0.009%, about 0.01%, about 0.011%, about 0.012%, about 0.013%,about 0.014%, about 0.015%, about 0.016%, about 0.017%, about 0.018%,about 0.019%, or about 0.02%. In some embodiments, the concentration ofcalcium hydroxyapatite is about 0.05%, about 0.1%, about 0.15%, about0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%,about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%,about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about1.6%, about 1.65%, about 1.7%, about 1.75%, about 1.8%, about 1.85%,about 1.9%, about 1.95%, or about 2%. In some embodiments, the organiccompound comprises an amino acid selected from the group consisting ofglycine, L-proline, alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In some embodiments, the invention relates to a tissue filler comprisingHA, wherein the HA is obtained from Streptococcus bacteria, or fromBacillus subtilis bacteria.

In one embodiment, the invention relates to a biocompatible tissuefiller comprising: a glycosaminoglycan selected from the groupconsisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC),starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthangum, chitosan, pectin, agar, carrageenan, and guar gum; and ananesthetic agent; wherein a portion of the glycosaminoglycan iscross-linked by cross-linking moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol; andwherein cross-linking is obtained using a cross-linking agent, across-linking precursor, or an activating agent. In some embodiments,the anesthetic agent is lidocaine. In some embodiments, theconcentration of anesthetic agent in the tissue filler is from about0.001% to about 5%. In some embodiments, the concentration of lidocainein the tissue filler is about 0.3%.

In one embodiment, the invention relates to a biocompatible tissuefiller comprising: a glycosaminoglycan selected from the groupconsisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC),starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthangum, chitosan, pectin, agar, carrageenan, and guar gum; and ananesthetic agent; wherein a portion of the glycosaminoglycan iscross-linked by cross-linking moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol; andwherein cross-linking is obtained using a cross-linking agent, across-linking precursor, or an activating agent; wherein the tissuefiller is a gel. In some embodiments, the tissue filler is a hydrogel.In some embodiments, the tissue filler further comprises water. In someembodiments, the total concentration of HA in the tissue filler is fromabout 10 mg/niL to about 50 mg/mL. In some embodiments, the totalconcentration of HA in the tissue filler is about 15 mg/mL, about 16mg/mL, 17 mg/mL, about 18 mg/mL, about 19 mg/mL, about 20 mg/mL, about21 mg/mL, about 22 mg/mL, about 23 mg/mL, about 24 mg/mL, about 25mg/mL, about 26 mg/mL, about 27 mg/mL, about 28 mg/mL, about 29 mg/mL,or about 30 mg/mL. In some embodiments, the concentration of crosslinked HA in the tissue filler is from about 10 mg/mL to about 50 mg/mL.In some embodiments, the concentration of cross linked HA in the tissuefiller is about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18mg/mL, about 19 mg/mL, about 20 mg/mL, about 21 mg/mL, about 22 mg/mL,about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26 mg/mL, about 27mg/mL, about 28 mg/mL, about 29 mg/mL, or about 30 mg/mL.

In one embodiment, the invention relates to a biocompatible tissuefiller comprising: a glycosaminoglycan selected from the groupconsisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC),starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthangum, chitosan, pectin, agar, carrageenan, and guar gum; and ananesthetic agent; wherein a portion of the glycosaminoglycan iscross-linked by cross-linking moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol; andwherein cross-linking is obtained using a cross-linking agent, across-linking precursor, or an activating agent; the tissue fillercomprising silk protein or silk protein fragments (SPF). In someembodiments, the silk protein is silk fibroin. In some embodiments, thesilk protein is silk fibroin substantially devoid of sericin. In someembodiments, the SPF have an average weight average molecular weightranging from about 1 kDa to about 250 kDa. In some embodiments, the SPFhave an average weight average molecular weight ranging from about 5 kDato about 150 kDa. In some embodiments, the SPF have an average weightaverage molecular weight ranging from about 6 kDa to about 17 kDa. Insome embodiments, the SPF have an average weight average molecularweight ranging from about 17 kDa to about 39 kDa. In some embodiments,the SPF have an average weight average molecular weight ranging fromabout 39 kDa to about 80 kDa. In some embodiments, the SPF have lowmolecular weight. In some embodiments, the SPF have medium molecularweight. In some embodiments, the SPF have high molecular weight. In someembodiments, the silk protein fragments (SPF) have a polydispersity ofbetween about 1.5 and about 3.0. In some embodiments, the SPF have adegree of crystallinity of up to 60%. In some embodiments, a portion ofthe SPF are cross-linked. In some embodiments, the % w/w amount ofcross-linked SPF relative to the total amount of SPF is about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%,about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%,about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about35%, about 36%, about 37%, about 38%, about 39%, about 400%, about 41%,about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%,about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%,about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%,about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%,about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, orabout 100%. In some embodiments, the degree of cross-linking of thecross-linked SPF is between about 1% and about 100%. In someembodiments, the degree of cross-linking of the cross-linked SPF isabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%,about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%,about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%,about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%,about 99%, or about 100%. In some embodiments, the degree ofcross-linking of the cross-linked SPF is between about 1% and about 15%.In some embodiments, the degree of cross-linking of the cross-linked SPFis one or more of about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, and about 15%.

In one embodiment, the invention relates to a biocompatible tissuefiller comprising: a glycosaminoglycan selected from the groupconsisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC),starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthangum, chitosan, pectin, agar, carrageenan, and guar gum; and ananesthetic agent; wherein a portion of the glycosaminoglycan iscross-linked by cross-linking moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol; andwherein cross-linking is obtained using a cross-linking agent, across-linking precursor, or an activating agent; the tissue fillercomprising silk protein or silk protein fragments (SPF), wherein aportion of the SPF are cross-linked. In some embodiments, thecross-linked SPF comprises a cross-linking moiety comprising an alkaneor alkyl chain, and/or an ether group. In some embodiments, thecross-linked SPF comprises a cross-linking moiety comprising apolyethylene glycol (PEG) chain. In some embodiments, the cross-linkedSPF comprises a cross-linking moiety comprising a secondary alcohol. Insome embodiments, cross-linking is obtained using a cross-linking agent,a cross-linking precursor, or an activating agent. In some embodiments,the cross-linking agent and/or the cross-linking precursor comprises anepoxy group. In some embodiments, cross-linking is obtained using across-linking agent, a cross-linking precursor, or an activating agentselected from the group consisting of a polyepoxy linker, a diepoxylinker, a polyepoxy-PEG, a diepoxy-PEG, a polyglycidyl-PEG, adiglycidyl-PEG, a poly acrylate PEG, a diacrylate PEG,1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, divinylsulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light,glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE),1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritoltetraglycidyl ether (PETGE), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, a carbodiimide, and anycombinations thereof. In some embodiments, cross-linking is obtainedusing a polyfunctional epoxy compound selected from the group consistingof 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidylether (EGDGE), 1,6-hexanediol diglycidyl ether, polyethylene glycoldiglycidyl ether, polypropylene glycol diglycidyl ether,polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidylether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether,glycerol polyglycidyl ether, tri-methylolpropane polyglycidyl ether,pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether. Insome embodiments, cross-linking is obtained using a cross-linking agentand/or a cross-linking precursor selected from the group consisting ofpolyethylene glycol diglycidyl ether, diepoxy PEG, PEG diglycidyl ether,polyoxyethylene bis-glycidyl ether, PEGDE, and PEGDGE. In someembodiments, cross-linking is obtained using polyethylene glycoldiglycidyl ether having an average M_(n) of about 500, about 1000, about2000, or about 6000. In some embodiments, cross-linking is obtainedusing polyethylene glycol diglycidyl ether having from 2 to 25 ethyleneglycol groups. In some embodiments, cross-linking is obtained using across-linking agent and/or a cross-linking precursor selected from thegroup consisting of a polyepoxy silk fibroin linker, a diepoxy silkfibroin linker, a polyepoxy silk fibroin fragment linker, a diepoxy silkfibroin fragment linker, a polyglycidyl silk fibroin linker, adiglycidyl silk fibroin linker, a polyglycidyl silk fibroin fragmentlinker, and a diglycidyl silk fibroin fragment linker. In someembodiments, a portion of SPF is cross linked to HA. In someembodiments, a portion of the SPF are cross-linked to SPF. In someembodiments, the tissue filler is a gel. In some embodiments, the tissuefiller is a hydrogel. In some embodiments, the tissue filler furthercomprises water. In some embodiments, the total concentration of SPF inthe tissue filler is from about 0.1 mg/mL to about 15 mg/mL. In someembodiments, the total concentration of SPF in the tissue filler isabout 0.1 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL,about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 8.5mg/mL, about 9 mg/mL, about 9.5 mg/mL, about 10 mg/mL, about 10.5 mg/mL,about 11 mg/mL, about 11.5 mg/mL, about 12 mg/mL, about 12.5 mg/mL,about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 14.5 mg/mL, orabout 15 mg/mL. In some embodiments, the concentration of cross linkedSPF in the tissue filler is from about 0.1 mg/mL to about 15 mg/mL. Insome embodiments, the concentration of cross linked SPF in the tissuefiller is about 0.1 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 1.5mg/mL, about 2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL,about 4 mg/mL, about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6mg/mL, about 6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL,about 8.5 mg/mL, about 9 mg/mL, about 9.5 mg/mL, about 10 mg/mL, about10.5 mg/mL, about 11 mg/mL, about 11.5 mg/mL, about 12 mg/mL, about 12.5mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 14.5mg/mL, or about 15 mg/mL.

In one embodiment, the invention relates to a biocompatible tissuefiller comprising: a glycosaminoglycan selected from the groupconsisting of hyaluronic acid (HA), carboxymethyl cellulose (CMC),starch, alginate, chondroitin-4-sulfate, chondroitin-6-sulfate, xanthangum, chitosan, pectin, agar, carrageenan, and guar gum; and ananesthetic agent; wherein a portion of the glycosaminoglycan iscross-linked by cross-linking moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol; andwherein cross-linking is obtained using a cross-linking agent, across-linking precursor, or an activating agent; the tissue filleroptionally comprising silk protein or silk protein fragments (SPF),wherein a portion of the SPF are cross-linked. In some embodiments, thetissue filler is a dermal filler. In some embodiments, the tissue filleris biodegradable. In some embodiments, the tissue filler is injectable.In some embodiments, the tissue filler has a storage modulus (G′) offrom about 25 Pa to about 1500 Pa. In some embodiments, the tissuefiller has a storage modulus (G′) of about 25 Pa, about 26 Pa, about 27Pa, about 28 Pa, about 29 Pa, about 30 Pa, about 31 Pa, about 32 Pa,about 33 Pa, about 34 Pa, about 35 Pa, about 36 Pa, about 37 Pa, about38 Pa, about 39 Pa, about 40 Pa, about 41 Pa, about 42 Pa, about 43 Pa,about 44 Pa, about 45 Pa, about 46 Pa, about 47 Pa, about 48 Pa, about49 Pa, about 50 Pa, about 51 Pa, about 52 Pa, about 53 Pa, about 54 Pa,about 55 Pa, about 56 Pa, about 57 Pa, about 58 Pa, about 59 Pa, about60 Pa, about 61 Pa, about 62 Pa, about 63 Pa, about 64 Pa, about 65 Pa,about 66 Pa, about 67 Pa, about 68 Pa, about 69 Pa, about 70 Pa, about71 Pa, about 72 Pa, about 73 Pa, about 74 Pa, about 75 Pa, about 76 Pa,about 77 Pa, about 78 Pa, about 79 Pa, about 80 Pa, about 81 Pa, about82 Pa, about 83 Pa, about 84 Pa, about 85 Pa, about 86 Pa, about 87 Pa,about 88 Pa, about 89 Pa, about 90 Pa, about 91 Pa, about 92 Pa, about93 Pa, about 94 Pa, about 95 Pa, about 96 Pa, about 97 Pa, about 98 Pa,about 99 Pa, about 100 Pa, about 101 Pa, about 102 Pa, about 103 Pa,about 104 Pa, about 105 Pa, about 106 Pa, about 107 Pa, about 108 Pa,about 109 Pa, about 110 Pa, about 111 Pa, about 112 Pa, about 113 Pa,about 114 Pa, about 115 Pa, about 116 Pa, about 117 Pa, about 118 Pa,about 119 Pa, about 120 Pa, about 121 Pa, about 122 Pa, about 123 Pa,about 124 Pa, or about 125 Pa. In some embodiments, herein G′ ismeasured by means of an oscillatory stress of about 0.1 to about 10 Hz.In some embodiments, G′ is measured by means of an oscillatory stress ofabout 1 Hz. In some embodiments, G′ is measured by means of anoscillatory stress of about 5 Hz. In some embodiments, G′ is measured bymeans of an oscillatory stress of about 10 Hz. In some embodiments, thetissue filler has a complex viscosity from about 1 Pa·s to about 10Pa·s. In some embodiments, the tissue filler has a complex viscosity ofabout 1 Pa·s, about 1.5 Pa·s, about 2 Pa·s, about 2.5 Pa·s, about 3Pa·s, about 3.5 Pa·s, about 4 Pa·s, about 4.5 Pa·s, about 5 Pa·s, about5.5 Pa·s, about 6 Pa·s, about 6.5 Pa·s, about 7 Pa·s, about 7.5 Pa·s,about 8 Pa·s, about 8.5 Pa·s, about 9 Pa·s, about 9.5 Pa·s, or about 10Pa·s. In some embodiments, the complex viscosity is measured by means ofan oscillatory stress of about 0.1 to about 10 Hz. In some embodiments,the complex viscosity is measured by means of an oscillatory stress ofabout 1 Hz. In some embodiments, the complex viscosity is measured bymeans of an oscillatory stress of about 5 Hz.

In one embodiment, the invention relates to a method of treating acondition in a subject in need thereof, and/or a method of cosmetictreatment in a subject in need thereof, the method comprisingadministering to the subject a therapeutically effective amount of abiocompatible tissue filler comprising: a glycosaminoglycan selectedfrom the group consisting of hyaluronic acid (HA), carboxymethylcellulose (CMC), starch, alginate, chondroitin-4-sulfate,chondroitin-6-sulfate, xanthan gum, chitosan, pectin, agar, carrageenan,and guar gum; and an anesthetic agent; wherein a portion of theglycosaminoglycan is cross-linked by cross-linking moieties comprisingone or more of an alkane or alkyl chain, an ether group, and a secondaryalcohol; and wherein cross-linking is obtained using a cross-linkingagent, a cross-linking precursor, or an activating agent; the tissuefiller optionally comprising silk protein or silk protein fragments(SPF), wherein a portion of the SPF are cross-linked. In someembodiments, the condition is a skin condition. In some embodiments, theskin condition is selected from the group consisting of skindehydration, lack of skin elasticity, skin roughness, lack of skintautness, a skin stretch line, a skin stretch mark, skin paleness, adermal divot, a sunken cheek, a thin lip, a retro-orbital defect, afacial fold, and a wrinkle. In some embodiments the tissue filler isadministered into a dermal region of the subject. In some embodiments,the method is an augmentation, a reconstruction, treating a disease,treating a disorder, correcting a defect or imperfection of a body part,region or area. In some embodiments, the method is a facialaugmentation, a facial reconstruction, treating a facial disease,treating a facial disorder, treating a facial defect, or treating afacial imperfection. In some embodiments, the tissue filler resistsbiodegradation, bioerosion, bioabsorption, and/or bioresorption, for atleast about 3 days, about 7 days, about 14 days, about 21 days, about 28days, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, or about 6 months. In some embodiments, administrationof the tissue filler to the subject results in a reduced inflammatoryresponse compared to the inflammatory response induced by a controltissue filler comprising a polysaccharide and lidocaine, wherein thecontrol tissue filler does not include silk protein fragments (SPF). Insome embodiments, administration of the tissue filler to the subjectresults in increased collagen production compared to the collagenproduction induced by a control tissue filler comprising apolysaccharide and lidocaine, wherein the control tissue filler does notinclude silk protein fragments (SPF).

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and a polysaccharide. In someembodiments, the polysaccharide is hyaluronic acid (HA). In anembodiment, the invention includes tissue fillers that may be preparedfrom silk and hyaluronic acid.

In some embodiments, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) with an average molecularweight ranging from about 1 kDa to about 250 kDa. In some embodiments,the invention relates to a biocompatible tissue filler including silkprotein fragments (SPF) with an average molecular weight ranging fromabout 5 kDa to about 150 kDa. In some embodiments, the SPF have anaverage molecular weight ranging from about 6 kDa to about 17 kDa. Insome embodiments, the SPF have an average molecular weight ranging fromabout 17 kDa to about 39 kDa. In some embodiments, the SPF have anaverage molecular weight ranging from about 39 kDa to about 80 kDa. Insome embodiments, the SPF have an average molecular weight ranging fromabout 80 kDa to about 150 kDa.

In some embodiments, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) which are up to about 0%to 100% cross-linked with SPF. In some embodiments, the SPF werecross-linked to SPF using cross-linking agents such as BDDE, or one ofthe other cross-linking agents described herein. In some embodiments,the degree of cross-linking is up to about 100%.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and hyaluronic acid (HA), wherein up toabout 0% to 100% of the SPF are cross-linked to SPF, and the SPF werecross-linked to SPF using a cross-linking agent such as BDDE, or one ofthe other cross-linking agents described herein, and the SPF degree ofcross-linking is up to about 100%.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and hyaluronic acid (HA), wherein up to100% of HA is cross-linked to HA using a cross-linking agent such asBDDE, or one of the other cross-linking agents described herein. In someembodiments, up to about 100% of the SPF are cross-linked to SPF,wherein the SPF were cross-linked to SPF using a cross-linking agentsuch as BDDE, or one of the other cross-linking agents described herein,and the SPF degree of cross-linking is up to about 100%.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and hyaluronic acid (HA), wherein 0% to100% of HA is non-cross-linked. In some embodiments, up to about 100% ofthe SPF are cross-linked, wherein the SPF were cross-linked using across-linking agent such as BDDE, or one of the other cross-linkingagents described herein, and the SPF degree of cross-linking is up toabout 100%. In some embodiments, all of the HA is non-cross-linked.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and hyaluronic acid (HA), wherein 0% to100% of SPF is cross-linked to HA. In some embodiments, the SPF and HAwere cross-linked using a cross-linking agent such as BDDE, or one ofthe cross-linking agents described herein. In some embodiments, thedegree of SPF-HA cross-linking is up to about 100%. In some embodiments,up to 100% of HA is cross-linked to HA. In some embodiments, HA wascross-linked to HA using a cross-linking agent such as BDDE, or one ofthe cross-linking agents described herein. In some embodiments, at least0.1% of HA is non-cross-linked. In some embodiments, all of the HA isnon-cross-linked.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and hyaluronic acid (HA), wherein atleast 0.1% of HA is non-cross-linked. In some embodiments, up to about100% of the SPF are cross-linked, wherein the SPF were cross-linkedusing a cross-linking agent such as BDDE, or one of the othercross-linking agents described herein, and the SPF degree ofcross-linking is up to about 100%. In some embodiments, all of the HA isnon-cross-linked.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and hyaluronic acid (HA), wherein atleast 0.1% of SPF is cross-linked to HA. In some embodiments, the SPFand HA were cross-linked using a cross-linking agent such as BDDE, orone of the cross-linking agents described herein. In some embodiments,the degree of SPF-HA cross-linking is up to about 100%. In someembodiments, up to 100% of HA is cross-linked to HA. In someembodiments, HA was cross-linked to HA using a cross-linking agent suchas BDDE, or one of the cross-linking agents described herein. In someembodiments, at least 0.1% of HA is non-cross-linked. In someembodiments, all of the HA is non-cross-linked.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and a polysaccharide, wherein the SPFare substantially devoid of sericin.

In one embodiment, the invention relates to a biocompatible gel tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and a polysaccharide.

In one embodiment, the invention relates to a biocompatible hydrogeltissue filler including silk protein fragments (SPF) having apolydispersity of between about 1.5 and about 3.0, and a polysaccharide

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, a polysaccharide, and water.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and a polysaccharide, wherein SPF havea degree of crystallinity of about 0% to about 60%.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and a polysaccharide, and furtherincluding an active agent. In some embodiments, the active agent can bean enzyme inhibitor, an anesthetic agent, a medicinal neurotoxin, anantioxidant, an anti-infective agent, vasodilators, a reflective agent,an anti-inflammatory agent, an ultraviolet (UV) light blocking agent, adye, a hormone, an immunosuppressant, or an anti-inflammatory agent. Inone embodiment, the anesthetic agent is lidocaine.

In one embodiment, the invention relates to an injectable biocompatibletissue filler including silk protein fragments (SPF) having apolydispersity of between about 1.5 and about 3.0, and a polysaccharide.

In one embodiment, the invention relates to a biocompatible tissuefiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, and a polysaccharide. In someembodiments, G′ is measured by means of an oscillatory stress of about0.1 to about 10 Hz. In one embodiment, G′ is measured by means of anoscillatory stress of about 1 Hz.

In one embodiment, the invention relates to a method of making abiocompatible tissue filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the method including providing an SPF solution, andadding to the solution a gelation enhancer, which may be any protondonating species.

In one embodiment, the invention relates to a method of making abiocompatible tissue filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the method including providing an SPF solution, andsubjecting the solution to mechanical excitation.

In one embodiment, the invention relates to a method of treating acondition in a subject in need thereof, the method includingadministering to the subject a therapeutically effective amount of abiocompatible tissue filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide. In some embodiments, the condition is a skin condition.In some embodiments, the skin condition can be skin dehydration, lack ofskin elasticity, skin roughness, lack of skin tautness, a skin stretchline, a skin stretch mark, skin paleness, a dermal divot, a sunkencheek, sunken temple, a thin lip, a retro-orbital defect, a facial fold,or a wrinkle.

In one embodiment, the invention relates to a method of cosmetictreatment in a subject in need thereof, the method includingadministering to the subject an effective amount of a biocompatibletissue filler including silk protein fragments (SPF) having apolydispersity of between about 1.5 and about 3.0, and a polysaccharide.

In some embodiments, the methods of the invention include administeringa biocompatible tissue filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, into a dermal region of a subject.

In one embodiment, a method of the invention including administering abiocompatible tissue filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, can be an augmentation, a reconstruction, treating adisease, treating a disorder, correcting a defect or imperfection of abody part, region or area.

In one embodiment, a method of the invention including administering abiocompatible tissue filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, can be a facial augmentation, a facial reconstruction,treating a facial disease, treating a facial disorder, treating a facialdefect, or treating a facial imperfection.

In one embodiment, a biocompatible tissue filler including silk proteinfragments (SPF) having a polydispersity of between about 1.5 and about3.0, and a polysaccharide, administered according to a method of theinvention, resists biodegradation, bioabsorption, and/or bioresorption,for at least about 3 days after administration.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,the tissue filler further includes cross-linking moieties, e.g., epoxyderived cross-linking moieties. In some embodiments, a portion ofcross-linking is auto-cross-linking. In some embodiments, the portion ofcross-linked SPF is up to about 100%. In some embodiments, the portionof cross-linked polysaccharide is up to about 100%. In some embodiments,the polysaccharide is hyaluronic acid (HA). In some embodiments, the SPFare substantially devoid of sericin. In some embodiments, tissue fillerfurther comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,the tissue filler further includes cross-linking moieties, e.g., epoxyderived cross-linking moieties. In some embodiments, a portion ofcross-linking is auto-cross-linking. In some embodiments, the portion ofcross-linked SPF is up to about 100%. In some embodiments, the portionof cross-linked polysaccharide is up to about 100%. In some embodiments,the polysaccharide is hyaluronic acid (HA). In some embodiments, the SPFare substantially devoid of sericin. In some embodiments, tissue fillerfurther comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the SPF are substantially devoid of sericin.In some embodiments, tissue filler further comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the SPF are substantially devoid of sericin.In some embodiments, tissue filler further comprises water.

In some embodiments, the % w/w amount of cross-linked SPF relative tothe total amount of SPF is up to about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100%.

In some embodiments, the degree of cross-linking of SPF is up to about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%,about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100%.

In some embodiments, the % w/w amount of cross-linked HA relative to thetotal amount of HA is up to about 1%, about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%,about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%,about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%,about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%,about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%,about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%,about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about96%, about 97%, about 98%, about 99%, or about 100%.

In some embodiments, the degree of cross-linking of HA is up to about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%,about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%,about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%,about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%,about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%,about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%,about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%,about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about99%, or about 100%.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, cross-linking is obtained using across-linking agent, a cross-linking precursor, or an activating agent.In some embodiments, the cross-linking agent and/or the cross-linkingprecursor comprise an epoxy group. In some embodiments, the SPF aresubstantially devoid of sericin.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, cross-linking is obtained using across-linking agent, a cross-linking precursor, or an activating agent.In some embodiments, the cross-linking agent and/or the cross-linkingprecursor comprise an epoxy group. In some embodiments, the SPF aresubstantially devoid of sericin.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, cross-linking is obtained using across-linking agent, a cross-linking precursor, or an activating agentselected from the group consisting of 1,4-bis(2,3-epoxypropoxy)butane,1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanedioldiglycidyl ether (BDDE), UV light, glutaraldehyde,1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE),adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS),hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, acarbodiimide, and any combinations thereof. In some embodiments, the SPFare substantially devoid of sericin.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, cross-linking is obtained using across-linking agent, a cross-linking precursor, or an activating agentselected from the group consisting of 1,4-bis(2,3-epoxypropoxy)butane,1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanedioldiglycidyl ether (BDDE), UV light, glutaraldehyde,1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE),adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS),hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, acarbodiimude, and any combinations thereof. In some embodiments, the SPFare substantially devoid of sericin.

In one embodiment, the invention relates to a biocompatible tissuefiller gel, e.g., a dermal filler gel, including silk protein fragments(SPF) having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the gel further comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller gel, e.g., a dermal filler gel, including silk protein fragments(SPF) having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the gel further comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller hydrogel, e.g., a dermal filler hydrogel, including silk proteinfragments (SPF) having a polydispersity of between about 1.5 and about3.0, and a polysaccharide, the SPF having an average weight averagemolecular weight ranging from about 1 kDa to about 250 kDa, about 5 kDato about 150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa toabout 39 kDa, or from about 39 kDa to about 80 kDa. In some embodiments,the tissue filler is biodegradable. In some embodiments, a portion ofSPF are cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the hydrogel further comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller hydrogel, e.g., a dermal filler hydrogel, including silk proteinfragments (SPF) having a polydispersity of between about 1.5 and about3.0, and a polysaccharide, the SPF having low molecular weight, mediummolecular weight, and/or high molecular weight. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the hydrogel further comprises water.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the SPF have a degree of crystallinity of upto about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%,about 60%, or more than 60%.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the SPF have a degree of crystallinity of upto about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%,about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%,about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%,about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%,about 60%, or more than 60%.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the tissue filler further comprises an activeagent. In some embodiments, the active agent is selected from the groupconsisting of an enzyme inhibitor, an anesthetic agent, a medicinalneurotoxin, an antioxidant, an anti-infective agents, ananti-inflammatory agent, an ultraviolet (UV) light blocking agent, adye, a hormone, an immunosuppressant, and an anti-inflammatory agent. Insome embodiments, the anesthetic agent is lidocaine.

In one embodiment, the invention relates to a biocompatible tissuefiller, e.g., a dermal filler, including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the tissue filler further comprises an activeagent. In some embodiments, the active agent is selected from the groupconsisting of an enzyme inhibitor, an anesthetic agent, a medicinalneurotoxin, an antioxidant, an anti-infective agent, ananti-inflammatory agent, an ultraviolet (UV) light blocking agent, adye, a hormone, an immunosuppressant, and an anti-inflammatory agent. Insome embodiments, the anesthetic agent is lidocaine.

In one embodiment, the invention relates to a biocompatible injectabletissue filler, e.g., an injectable dermal filler, including silk proteinfragments (SPF) having a polydispersity of between about 1.5 and about3.0, and a polysaccharide, the SPF having an average weight averagemolecular weight ranging from about 1 kDa to about 250 kDa, about 5 kDato about 150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa toabout 39 kDa, or from about 39 kDa to about 80 kDa. In some embodiments,the tissue filler is biodegradable. In some embodiments, a portion ofSPF are cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA).

In one embodiment, the invention relates to a biocompatible injectabletissue filler, e.g., an injectable dermal filler, including silk proteinfragments (SPF) having a polydispersity of between about 1.5 and about3.0, and a polysaccharide, the SPF having low molecular weight, mediummolecular weight, and/or high molecular weight. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA).

In one embodiment, the invention relates to a biocompatible tissuefiller having a storage modulus (G′) of from about 50 Pa to about 1500Pa, e.g., a dermal filler having a storage modulus (G′) of from about 50Pa to about 1500 Pa, the filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 5 kDa to about 150 kDa, from about 6 kDa toabout 17 kDa, from about 17 kDa to about 39 kDa, or from about 39 kDa toabout 80 kDa. In some embodiments, the tissue filler is biodegradable.In some embodiments, a portion of SPF are cross-linked. In someembodiments, a portion of the SPF are cross-linked to polysaccharide. Insome embodiments, a portion of the SPF are cross-linked to SPF. In someembodiments, a portion of the polysaccharide is cross-linked topolysaccharide. In some embodiments, cross-linking includes chemicalbond cross-linking. In some embodiments, a portion of cross-linking iszero-length cross-linking. In some embodiments, a portion ofcross-linking is auto-cross-linking. In some embodiments, the portion ofcross-linked SPF is up to about 100%. In some embodiments, the portionof cross-linked polysaccharide is up to about 100%. In some embodiments,the polysaccharide is hyaluronic acid (HA). In some embodiments, G′ ismeasured by means of an oscillatory stress of about 0.1 to about 10 Hz.In some embodiments, G′ is measured by means of an oscillatory stress ofabout 1 Hz.

In one embodiment, the invention relates to a biocompatible tissuefiller having a storage modulus (G′) of from about 50 Pa to about 1500Pa, e.g., a dermal filler having a storage modulus (G′) of from about 50Pa to about 1500 Pa, the filler including silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, or high molecular weight. In some embodiments, the tissue filleris biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,cross-linking includes chemical bond cross-linking. In some embodiments,a portion of cross-linking is zero-length cross-linking. In someembodiments, a portion of cross-linking is auto-cross-linking. In someembodiments, the portion of cross-linked SPF is up to about 100%. Insome embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, G′ is measured by means of an oscillatorystress of about 0.1 to about 10 Hz. In some embodiments, G′ is measuredby means of an oscillatory stress of about 1 Hz.

In some embodiments, the invention relates to a method of making abiocompatible tissue filler. e.g., a dermal filler, including silkprotein fragments (SPF) having a polydispersity of between about 1.5 andabout 3.0, and a polysaccharide, the method including providing acomposition comprising SPF and a polysaccharide, and adding to thesolution a cross-linking agent, a cross-linking precursor, an activatingagent, or a gelation enhancer, the SPF having an average weight averagemolecular weight ranging from about 1 kDa to about 250 kDa, about 5 kDato about 150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa toabout 39 kDa, or from about 39 kDa to about 80 kDa. In some embodiments,the tissue filler is biodegradable. In some embodiments, a portion ofSPF are cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,the tissue filler further includes cross-linking moieties, e.g., epoxyderived cross-linking moieties. In some embodiments, a portion ofcross-linking is auto-cross-linking. In some embodiments, the portion ofcross-linked SPF is up to about 100%. In some embodiments, the portionof cross-linked polysaccharide is up to about 100%. In some embodiments,the polysaccharide is hyaluronic acid (HA). In some embodiments, the SPFare substantially devoid of sericin. In some embodiments, the tissuefiller further comprises water.

In some embodiments, the invention relates to a method of making abiocompatible tissue filler, e.g., a dermal filler, including silkprotein fragments (SPF) having a polydispersity of between about 1.5 andabout 3.0, and a polysaccharide, the method including providing acomposition comprising SPF and a polysaccharide, and adding to thesolution a cross-linking agent, a cross-linking precursor, an activatingagent, or a gelation enhancer, the SPF having low molecular weight,medium molecular weight, and/or high molecular weight. In someembodiments, the tissue filler is biodegradable. In some embodiments, aportion of SPF are cross-linked. In some embodiments, a portion of theSPF are cross-linked to polysaccharide. In some embodiments, a portionof the SPF are cross-linked to SPF. In some embodiments, a portion ofthe polysaccharide is cross-linked to polysaccharide. In someembodiments, the tissue filler further includes cross-linking moieties,e.g., epoxy derived cross-linking moieties. In some embodiments, aportion of cross-linking is auto-cross-linking. In some embodiments, theportion of cross-linked SPF is up to about 100%. In some embodiments,the portion of cross-linked polysaccharide is up to about 100%. In someembodiments, the polysaccharide is hyaluronic acid (HA). In someembodiments, the SPF are substantially devoid of sericin. In someembodiments, tissue filler further comprises water.

In some embodiments, the invention relates to a method of treating acondition in a subject in need thereof, e.g., a skin condition, themethod comprising administering to the subject a therapeuticallyeffective amount of a biocompatible tissue filler, e.g., a dermalfiller, including silk protein fragments (SPF) having a polydispersityof between about 1.5 and about 3.0, and a polysaccharide, the SPF havingan average weight average molecular weight ranging from about 1 kDa toabout 250 kDa, about 5 kDa to about 150 kDa, from about 6 kDa to about17 kDa, from about 17 kDa to about 39 kDa, or from about 39 kDa to about80 kDa. In some embodiments, the tissue filler is biodegradable. In someembodiments, a portion of SPF are cross-linked. In some embodiments, aportion of the SPF are cross-linked to polysaccharide. In someembodiments, a portion of the SPF are cross-linked to SPF. In someembodiments, a portion of the polysaccharide is cross-linked topolysaccharide. In some embodiments, the tissue filler further includescross-linking moieties, e.g., epoxy derived cross-linking moieties. Insome embodiments, a portion of cross-linking is auto-cross-linking. Insome embodiments, the portion of cross-linked SPF is up to about 100%.In some embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the SPF are substantially devoid of sericin.In some embodiments, tissue filler further comprises water. In someembodiments, the skin condition is selected from the group consisting ofskin dehydration, lack of skin elasticity, skin roughness, lack of skintautness, a skin stretch line, a skin stretch mark, skin paleness, adermal divot, a sunken cheek, a thin lip, a retro-orbital defect, afacial fold, and a wrinkle. In some embodiments, the tissue filler isadministered into a dermal region of the subject. In some embodiments,the method is an augmentation, a reconstruction, treating a disease,treating a disorder, correcting a defect or imperfection of a body part,region or area. In some embodiments, the method is a facialaugmentation, a facial reconstruction, treating a facial disease,treating a facial disorder, treating a facial defect, or treating afacial imperfection. In some embodiments, the tissue filler resistsbiodegradation, bioerosion, bioabsorption, and/or bioresorption, for atleast about 3 days, about 7 days, about 14 days, about 21 days, about 28days, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, or about 6 months.

In some embodiments, the invention relates to a method of treating acondition in a subject in need thereof, e.g., a skin condition, themethod comprising administering to the subject a therapeuticallyeffective amount of a biocompatible tissue filler, e.g., a dermalfiller, including silk protein fragments (SPF) having a polydispersityof between about 1.5 and about 3.0, and a polysaccharide, the SPF havinglow molecular weight, medium molecular weight, and/or high molecularweight. In some embodiments, the tissue filler is biodegradable. In someembodiments, a portion of SPF are cross-linked. In some embodiments, aportion of the SPF are cross-linked to polysaccharide. In someembodiments, a portion of the SPF are cross-linked to SPF. In someembodiments, a portion of the polysaccharide is cross-linked topolysaccharide. In some embodiments, the tissue filler further includescross-linking moieties, e.g., epoxy derived cross-linking moieties. Insome embodiments, a portion of cross-linking is auto-cross-linking. Insome embodiments, the portion of cross-linked SPF is up to about 100%.In some embodiments, the portion of cross-linked polysaccharide is up toabout 100%. In some embodiments, the polysaccharide is hyaluronic acid(HA). In some embodiments, the SPF are substantially devoid of sericin.In some embodiments, tissue filler further comprises water. In someembodiments, the skin condition is selected from the group consisting ofskin dehydration, lack of skin elasticity, skin roughness, lack of skintautness, a skin stretch line, a skin stretch mark, skin paleness, adermal divot, a sunken cheek, a thin lip, a retro-orbital defect, afacial fold, and a wrinkle. In some embodiments, the tissue filler isadministered into a dermal region of the subject. In some embodiments,the method is an augmentation, a reconstruction, treating a disease,treating a disorder, correcting a defect or imperfection of a body part,region or area. In some embodiments, the method is a facialaugmentation, a facial reconstruction, treating a facial disease,treating a facial disorder, treating a facial defect, or treating afacial imperfection. In some embodiments, the tissue filler resistsbiodegradation, bioerosion, bioabsorption, and/or bioresorption, for atleast about 3 days, about 7 days, about 14 days, about 21 days, about 28days, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, or about 6 months.

In some embodiments, the invention relates to a method of cosmetictreatment in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a biocompatibletissue filler, e.g., a dermal filler, including silk protein fragments(SPF) having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, about 5 kDa to about150 kDa, from about 6 kDa to about 17 kDa, from about 17 kDa to about 39kDa, or from about 39 kDa to about 80 kDa. In some embodiments, thetissue filler is biodegradable. In some embodiments, a portion of SPFare cross-linked. In some embodiments, a portion of the SPF arecross-linked to polysaccharide. In some embodiments, a portion of theSPF are cross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,the tissue filler further includes cross-linking moieties, e.g., epoxyderived cross-linking moieties. In some embodiments, a portion ofcross-linking is auto-cross-linking. In some embodiments, the portion ofcross-linked SPF is up to about 100%. In some embodiments, the portionof cross-linked polysaccharide is up to about 100%. In some embodiments,the polysaccharide is hyaluronic acid (HA). In some embodiments, the SPFare substantially devoid of sericin. In some embodiments, tissue fillerfurther comprises water. In some embodiments, the tissue filler isadministered into a dermal region of the subject. In some embodiments,the method is an augmentation, a reconstruction, treating a disease,treating a disorder, correcting a defect or imperfection of a body part,region or area. In some embodiments, the method is a facialaugmentation, a facial reconstruction, treating a facial disease,treating a facial disorder, treating a facial defect, or treating afacial imperfection. In some embodiments, the tissue filler resistsbiodegradation, bioerosion, bioabsorption, and/or bioresorption, for atleast about 3 days, about 7 days, about 14 days, about 21 days, about 28days, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, or about 6 months.

In some embodiments, the invention relates to a method of cosmetictreatment in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a biocompatibletissue filler, e.g., a dermal filler, including silk protein fragments(SPF) having a polydispersity of between about 1.5 and about 3.0, and apolysaccharide, the SPF having low molecular weight, medium molecularweight, and/or high molecular weight. In some embodiments, the tissuefiller is biodegradable. In some embodiments, a portion of SPF arecross-linked. In some embodiments, a portion of the SPF are cross-linkedto polysaccharide. In some embodiments, a portion of the SPF arecross-linked to SPF. In some embodiments, a portion of thepolysaccharide is cross-linked to polysaccharide. In some embodiments,the tissue filler further includes cross-linking moieties, e.g., epoxyderived cross-linking moieties. In some embodiments, a portion ofcross-linking is auto-cross-linking. In some embodiments, the portion ofcross-linked SPF is up to about 100%. In some embodiments, the portionof cross-linked polysaccharide is up to about 100%. In some embodiments,the polysaccharide is hyaluronic acid (HA). In some embodiments, the SPFare substantially devoid of sericin. In some embodiments, tissue fillerfurther comprises water. In some embodiments, the tissue filler isadministered into a dermal region of the subject. In some embodiments,the method is an augmentation, a reconstruction, treating a disease,treating a disorder, correcting a defect or imperfection of a body part,region or area. In some embodiments, the method is a facialaugmentation, a facial reconstruction, treating a facial disease,treating a facial disorder, treating a facial defect, or treating afacial imperfection. In some embodiments, the tissue filler resistsbiodegradation, bioerosion, bioabsorption, and/or bioresorption, for atleast about 3 days, about 7 days, about 14 days, about 21 days, about 28days, about 1 month, about 2 months, about 3 months, about 4 months,about 5 months, or about 6 months.

In some embodiments, the invention relates to a biocompatible tissuefiller, comprising hyaluronic acid (HA) and an anesthetic agent, whereina portion of the HA is modified by one or more linker moietiescomprising one or more of an alkane or alkyl chain, an ether group, anda secondary alcohol, wherein the linker moieties are attached to the HAat one end of the linker. In some embodiments, modification is obtainedusing a cross-linking agent, a cross-linking precursor, or an activatingagent. In some embodiments, the HA in the tissue filler has a degree ofmodification (MoD) of about 10.0%, about 10.1%, about 10.2%, about10.3%, about 10.4%, about 10.5%, about 10.6%, about 10.7%, about 10.8%,about 10.9%, about 11.0%, about 11.1%, about 11.2%, about 11.3%, about11.4%, about 11.5%, about 11.6%, about 11.7%, about 11.8%, about 11.9%,about 12.0%, about 12.1%, about 12.2%, about 12.3%, about 12.4%, about12.5%, about 12.6%, about 12.7%, about 12.8%, about 12.9%, about 13.0%,about 13.1%, about 13.2%, about 13.3%, about 13.4%, about 13.5%, about13.6%, about 13.7%, about 13.8%, about 13.9%, about 14.0%, about 14.1%,about 14.2%, about 14.3%, about 14.4%, about 14.5%, about 14.6%, about14.7%, about 14.8%, about 14.9%, about 15.0%, about 15.1%, about 15.2%,about 15.3%, about 15.4%, about 15.5%, about 15.6%, about 15.7%, about15.8%, about 15.9%, about 16.0%, about 16.1%, about 16.2%, about 16.3%,about 16.4%, about 16.5%, about 16.6%, about 16.7%, about 16.8%, about16.9%, about 17.0%, about 17.1%, about 17.2%, about 17.3%, about 17.4%,about 17.5%, about 17.6%, about 17.7%, about 17.8%, about 17.9%, about18.0%, about 18.1%, about 18.2%, about 18.3%, about 18.4%, about 18.5%,about 18.6%, about 18.7%, about 18.8%, about 18.9%, about 19.0%, about19.1%, about 19.2%, about 19.3%, about 19.4%, about 19.5%, about 19.6%,about 19.7%, about 19.8%, about 19.9%, or about 20.0%. In someembodiments, the % w/w amount of modified HA relative to the totalamount of HA in the tissue filler is about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%,about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%,about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%,about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%,about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%,about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or about 100%.

In some embodiments, the modified HA includes cross-linked HA, whereinthe degree of cross-linking of the cross-linked HA is between about 1%and about 100%. In some embodiments, the degree of cross-linking of thecross-linked HA is about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%,about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%,about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%,about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%,about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%,about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100%. In some embodiments, thedegree of cross-linking of the cross-linked HA is between about 1% andabout 15%.

In some embodiments, the modified or cross-linked HA comprises a linkeror cross-linking moiety comprising a polyethylene glycol (PEG) chain. Insome embodiments, the cross-linking agent and/or the cross-linkingprecursor comprises an epoxy group. In some embodiments, modification orcross-linking is obtained using a cross-linking agent, a cross-linkingprecursor, or an activating agent selected from the group consisting ofa polyepoxy linker, a diepoxy linker, a polyepoxy-PEG, a diepoxy-PEG, apolyglycidyl-PEG, a diglycidyl-PEG, a poly acrylate PEG, a diacrylatePEG, 1,4-bis(2,3-epoxypropoxy)butane, 1,4-bisglycidyloxybutane, divinylsulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light,glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE),1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCDI), pentaerythritoltetraglycidyl ether (PETGE), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, a carbodiimide, and anycombinations thereof. In some embodiments, modification or cross-linkingis obtained using a polyfunctional epoxy compound selected from thegroup consisting of 1,4-butanediol diglycidyl ether (BDDE), ethyleneglycol diglycidyl ether (EGDGE), 1,6-hexanediol diglycidyl ether,polyethylene glycol diglycidyl ether, polypropylene glycol diglycidylether, polytetramethylene glycol diglycidyl ether, neopentyl glycoldiglycidyl ether, polyglycerol polyglycidyl ether, diglycerolpolyglycidyl ether, glycerol polyglycidyl ether, tri-methylolpropanepolyglycidyl ether, pentaerythritol polyglycidyl ether, and sorbitolpolyglycidyl ether. In some embodiments, modification or cross-linkingis obtained using a cross-linking agent and/or a cross-linking precursorselected from the group consisting of polyethylene glycol diglycidylether, diepoxy PEG, PEG diglycidyl ether, polyoxyethylene bis-glycidylether, PEGDE, and PEGDGE. In some embodiments, modification orcross-linking is obtained using polyethylene glycol diglycidyl etherhaving an average M_(n) of about 500, about 1000, about 2000, or about6000. In some embodiments, modification or cross-linking is obtainedusing polyethylene glycol diglycidyl ether having from about 2 to about25 ethylene glycol groups. In some embodiments, modification orcross-linking is obtained using a cross-linking agent and/or across-linking precursor selected from the group consisting of apolyepoxy silk fibroin linker, a diepoxy silk fibroin linker, apolyepoxy silk fibroin fragment linker, a diepoxy silk fibroin fragmentlinker, a polyglycidyl silk fibroin linker, a diglycidyl silk fibroinlinker, a polyglycidyl silk fibroin fragment linker, and a diglycidylsilk fibroin fragment linker.

In some embodiments, the tissue filler further includes an organiccompound and/or an inorganic compound. In some embodiments, theinorganic compound comprises calcium hydroxyapatite. In someembodiments, the calcium hydroxyapatite is formulated as particleshaving a diameter between about 1 μm and about 100 μm, between about 1μm and about 10 μm, between about 2 μm and about 12 μm, between about 3μm and about 10 μm, between about 4 μm and about 15 μm, between about 8μm and about 12 μm, between about 5 μm and about 10 μm, between about 6μm and about 12 μm, between about 7 μm and about 20 μm, between about 9μm and about 18 μm, or between about 10 μm and about μm. In someembodiments, the concentration of calcium hydroxyapatite is betweenabout 0.001% and about 5%. In some embodiments, the concentration ofcalcium hydroxyapatite is about 0.001%, about 0.002%, about 0.003%,about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%,about 0.009%, about 0.01%, about 0.011%, about 0.012%, about 0.013%,about 0.014%, about 0.015%, about 0.016%, about 0.017%, about 0.018%,about 0.019%, or about 0.02%. In some embodiments, the concentration ofcalcium hydroxyapatite is about 0.05%, about 0.1%, about 0.15%, about0.2%, about 0.25%, about 0.3%, about 0.35%, about 0.4%, about 0.45%,about 0.5%, about 0.55%, about 0.6%, about 0.65%, about 0.7%, about0.75%, about 0.8%, about 0.85%, about 0.9%, about 0.95%, about 1%, about1.05%, about 1.1%, about 1.15%, about 1.2%, about 1.25%, about 1.3%,about 1.35%, about 1.4%, about 1.45%, about 1.5%, about 1.55%, about1.6%, about 1.65%, about 1.7%, about 1.75%, about 1.8%, about 1.85%,about 1.9%, about 1.95%, or about 2%. In some embodiments, the organiccompound comprises an amino acid selected from the group consisting ofglycine, L-proline, alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, histidine, isoleucine, leucine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, and valine.

In some embodiments, the HA is obtained from Streptococcus bacteria, orfrom Bacillus subtilis bacteria. In some embodiments, the active agentis lidocaine. In some embodiments, the concentration of active agent inthe tissue filler is from about 0.001% to about 5%. In some embodiments,the concentration of lidocaine in the tissue filler is about 0.3%.

In some embodiments, the tissue filler disclosed herein is a gel. Insome embodiments, the tissue filler is a hydrogel. In some embodiments,the tissue filler further comprises water. In some embodiments, thetotal concentration of HA in the tissue filler is from about 10 mg/mL toabout 50 mg/mL. In some embodiments, the total concentration of HA inthe tissue filler is about 15 mg/mL, about 16 mg/mL, 17 mg/mL, about 18mg/mL, about 19 mg/mL, about 20 mg/mL, about 21 mg/mL, about 22 mg/mL,about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26 mg/mL, about 27mg/mL, about 28 mg/mL, about 29 mg/mL, or about 30 mg/mL. In someembodiments, the concentration of modified or cross linked HA in thetissue filler is from about 10 mg/mL to about 50 mg/mL. In someembodiments, the concentration of modified or cross linked HA in thetissue filler is about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about18 mg/mL, about 19 mg/mL, about 20 mg/mL, about 21 mg/mL, about 22mg/mL, about 23 mg/mL, about 24 mg/mL, about 25 mg/mL, about 26 mg/mL,about 27 mg/mL, about 28 mg/mL, about 29 mg/mL, or about 30 mg/mL.

In some embodiments, the tissue filler disclosed further includes silkprotein or silk protein fragments (SPF). In some embodiments, the silkprotein is silk fibroin. In some embodiments, the silk protein is silkfibroin substantially devoid of sericin. In some embodiments, the SPFhave an average weight average molecular weight ranging from about 1 kDato about 250 kDa. In some embodiments, the SPF have an average weightaverage molecular weight ranging from about 5 kDa to about 150 kDa. Insome embodiments, the SPF have an average weight average molecularweight ranging from about 6 kDa to about 17 kDa. In some embodiments,the SPF have an average weight average molecular weight ranging fromabout 17 kDa to about 39 kDa. In some embodiments, the SPF have anaverage weight average molecular weight ranging from about 39 kDa toabout 80 kDa. In some embodiments, the SPF have low molecular weight.

In some embodiments, the SPF have medium molecular weight. In someembodiments, the SPF have high molecular weight. In some embodiments,the silk protein fragments (SPF) have a polydispersity of between about1.5 and about 3.0. In some embodiments, the SPF have a degree ofcrystallinity of up to 60%.

In some embodiments, the invention relates to a tissue filler includingHA and SPF, wherein a portion of the SPF are modified or cross-linked.In some embodiments, the % w/w amount of modified or cross-linked SPFrelative to the total amount of SPF is about 1%, about 2%, about 3%,about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%,about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%,about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%,about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%,about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%,about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%,about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%,about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about95%, about 96%, about 97%, about 98%, about 99%, or about 100%. In someembodiments, the degree of modification or cross-linking of the modifiedor cross-linked SPF is between about 1% and about 100%. In someembodiments, the degree of modification or cross-linking of the modifiedor cross-linked SPF is about 1%, about 2%, about 3%, about 4%, about 5%,about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%,about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%,about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%,about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%,about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%,about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%,about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%,about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about97%, about 98%, about 99%, or about 100%. In some embodiments, thedegree of modification or cross-linking of the modified or cross-linkedSPF is between about 1% and about 15%. In some embodiments, the degreeof modification or cross-linking of the modified or cross-linked SPF isone or more of about 1%, about 2%, about 3%, about 4%, about 5%, about6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, and about 15%.

In some embodiments, the modified or cross-linked SPF comprises a linkeror cross-linking moiety comprising an alkane or alkyl chain, and/or anether group, wherein the linker or cross-linking moiety is attached tothe SPF at one end of the linker or cross-linking moiety. In someembodiments, the modified or cross-linked SPF comprises a linker orcross-linking moiety comprising a polyethylene glycol (PEG) chain. Insome embodiments, the modified or cross-linked SPF comprises a linker orcross-linking moiety comprising a secondary alcohol. In someembodiments, modification or cross-linking is obtained using amodification or cross-linking agent, a modification or cross-linkingprecursor, or an activating agent. In some embodiments, the modificationor cross-linking agent and/or the modification or cross-linkingprecursor comprises an epoxy group. In some embodiments, modification orcross-linking is obtained using a modification or cross-linking agent, amodification or cross-linking precursor, or an activating agent selectedfrom the group consisting of a polyepoxy linker, a diepoxy linker, apolyepoxy-PEG, a diepoxy-PEG, a polyglycidyl-PEG, a diglycidyl-PEG, apoly acrylate PEG, a diacrylate PEG, 1,4-bis(2,3-epoxypropoxy)butane,1,4-bisglycidyloxybutane, divinyl sulfone (DVS), 1,4-butanedioldiglycidyl ether (BDDE). UV light, glutaraldehyde,1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether (PETGE),adipic dihydrazide (ADH), bis(sulfosuccininidyl)suberate (BS),hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, acarbodiimide, and any combinations thereof. In some embodiments,modification or cross-linking is obtained using a polyfunctional epoxycompound selected from the group consisting of 1,4-butanediol diglycidylether (BDDE), ethylene glycol diglycidyl ether (EGDGE), 1,6-hexanedioldiglycidyl ether, polyethylene glycol diglycidyl ether, polypropyleneglycol diglycidyl ether, polytetramethylene glycol diglycidyl ether,neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether,diglycerol polyglycidyl ether, glycerol polyglycidyl ether,tri-methylolpropane polyglycidyl ether, pentaerythritol polyglycidylether, and sorbitol polyglycidyl ether.

In some embodiments, modification or cross-linking is obtained using amodification or cross-linking agent and/or a modification orcross-linking precursor selected from the group consisting ofpolyethylene glycol diglycidyl ether, diepoxy PEG, PEG diglycidyl ether,polyoxyethylene bis-glycidyl ether, PEGDE, and PEGDGE. In someembodiments, the modification or cross-linking is obtained usingpolyethylene glycol diglycidyl ether having an average M_(n) of about500, about 1000, about 2000, or about 6000. In some embodiments,modification or cross-linking is obtained using polyethylene glycoldiglycidyl ether having from about 2 to about 25 ethylene glycol groups.In some embodiments, modification or cross-linking is obtained using amodification or cross-linking agent and/or a modification orcross-linking precursor selected from the group consisting of apolyepoxy silk fibroin linker, a diepoxy silk fibroin linker, apolyepoxy silk fibroin fragment linker, a diepoxy silk fibroin fragmentlinker, a polyglycidyl silk fibroin linker, a diglycidyl silk fibroinlinker, a polyglycidyl silk fibroin fragment linker, and a diglycidylsilk fibroin fragment linker.

In some embodiments, the invention relates to a tissue filler includingHA and SPF, wherein a portion of SPF is cross linked to HA. In someembodiments, the invention relates to a tissue filler including HA andSPF, wherein a portion of the SPF are cross-linked to SPF. In someembodiments, the tissue filler is a gel. In some embodiments, the tissuefiller is a hydrogel. In some embodiments, the tissue filler furthercomprises water. In some embodiments, the total concentration of SPF inthe tissue filler is from about 0.1 mg/mL to about 15 mg/mL. In someembodiments, the total concentration of SPF in the tissue filler isabout 0.1 mg/mL, about 0.5 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about2 mg/mL, about 2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL,about 4.5 mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about6.5 mg/mL, about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 8.5mg/mL, about 9 mg/mL, about 9.5 mg/mL, about 10 mg/mL, about 10.5mg/niL, about 11 mg/mL, about 11.5 mg/mL, about 12 mg/mL, about 12.5mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 14.5mg/mL, or about 15 mg/mL. In some embodiments, the concentration ofmodified or cross linked SPF in the tissue filler is from about 0.1mg/mL to about 15 mg/mL. In some embodiments, the concentration ofmodified or cross linked SPF in the tissue filler is about 0.1 mg/mL,about 0.5 mg/mL, about 1 mg/mL, about 1.5 mg/mL, about 2 mg/mL, about2.5 mg/mL, about 3 mg/mL, about 3.5 mg/mL, about 4 mg/mL, about 4.5mg/mL, about 5 mg/mL, about 5.5 mg/mL, about 6 mg/mL, about 6.5 mg/mL,about 7 mg/mL, about 7.5 mg/mL, about 8 mg/mL, about 8.5 mg/mL, about 9mg/mL, about 9.5 mg/mL, about 10 mg/mL, about 10.5 mg/mL, about 11mg/mL, about 11.5 mng/mL, about 12 mg/mL, about 12.5 mg/mL, about 13mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 14.5 mg/mL, or about 15mg/mL.

In some embodiments, the invention relates to a tissue filler includingmodified or cross-linked HA, and/or modified or cross-linked SPF,wherein the tissue filler is a dermal filler. In some embodiments, thetissue filler is biodegradable. In some embodiments, the tissue filleris injectable. In some embodiments, the tissue filler has a storagemodulus (G) of from about 25 Pa to about 1500 Pa. In some embodiments,the tissue filler has a storage modulus (G′) of about 25 Pa, about 26Pa, about 27 Pa, about 28 Pa, about 29 Pa, about 30 Pa, about 31 Pa,about 32 Pa, about 33 Pa, about 34 Pa, about 35 Pa, about 36 Pa, about37 Pa, about 38 Pa, about 39 Pa, about 40 Pa, about 41 Pa, about 42 Pa,about 43 Pa, about 44 Pa, about 45 Pa, about 46 Pa, about 47 Pa, about48 Pa, about 49 Pa, about 50 Pa, about 51 Pa, about 52 Pa, about 53 Pa,about 54 Pa, about 55 Pa, about 56 Pa, about 57 Pa, about 58 Pa, about59 Pa, about 60 Pa, about 61 Pa, about 62 Pa, about 63 Pa, about 64 Pa,about 65 Pa, about 66 Pa, about 67 Pa, about 68 Pa, about 69 Pa, about70 Pa, about 71 Pa, about 72 Pa, about 73 Pa, about 74 Pa, about 75 Pa,about 76 Pa, about 77 Pa, about 78 Pa, about 79 Pa, about 80 Pa, about81 Pa, about 82 Pa, about 83 Pa, about 84 Pa, about 85 Pa, about 86 Pa,about 87 Pa, about 88 Pa, about 89 Pa, about 90 Pa, about 91 Pa, about92 Pa, about 93 Pa, about 94 Pa, about 95 Pa, about 96 Pa, about 97 Pa,about 98 Pa, about 99 Pa, about 100 Pa, about 101 Pa, about 102 Pa,about 103 Pa, about 104 Pa, about 105 Pa, about 106 Pa, about 107 Pa,about 108 Pa, about 109 Pa, about 110 Pa, about 111 Pa, about 112 Pa,about 113 Pa, about 114 Pa, about 115 Pa, about 116 Pa, about 117 Pa,about 118 Pa, about 119 Pa, about 120 Pa, about 121 Pa, about 122 Pa,about 123 Pa, about 124 Pa, or about 125 Pa. In some embodiments, G′ ismeasured by means of an oscillatory stress of about 0.1 to about 10 Hz.In some embodiments, G′ is measured by means of an oscillatory stress ofabout 1 Hz. In some embodiments, G′ is measured by means of anoscillatory stress of about 5 Hz. In some embodiments, G′ is measured bymeans of an oscillatory stress of about 10 Hz. In some embodiments, thetissue filler has a complex viscosity from about 1 Pa·s to about 10Pa·s. In some embodiments, the tissue filler has a complex viscosity ofabout 1 Pa·s, about 1.5 Pa·s, about 2 Pa·s, about 2.5 Pa·s, about 3Pa·s, about 3.5 Pa·s, about 4 Pa·s, about 4.5 Pa·s, about 5 Pa's, about5.5 Pa·s, about 6 Pa·s, about 6.5 Pa·s, about 7 Pa·s, about 7.5 Pa·s,about 8 Pa·s, about 8.5 Pa·s, about 9 Pa·s, about 9.5 Pa·s, or about 10Pa·s. In some embodiments, the complex viscosity is measured by means ofan oscillatory stress of about 0.1 to about 10 Hz. In some embodiments,the complex viscosity is measured by means of an oscillatory stress ofabout 1 Hz. In some embodiments, the complex viscosity is measured bymeans of an oscillatory stress of about 5 Hz.

In some embodiments, the invention relates to a method of treating acondition in a subject in need thereof, comprising administering to thesubject a therapeutically effective amount of a tissue filler includingmodified or cross-linked HA, and/or modified or cross-linked SPF. Insome embodiments, the condition is a skin condition. In someembodiments, the skin condition is selected from the group consisting ofskin dehydration, lack of skin elasticity, skin roughness, lack of skintautness, a skin stretch line, a skin stretch mark, skin paleness, adermal divot, a sunken cheek, a thin lip, a retro-orbital defect, afacial fold, and a wrinkle.

In some embodiments, the invention relates to a method of cosmetictreatment in a subject in need thereof, comprising administering to thesubject an effective amount of a tissue filler including modified orcross-linked HA, and/or modified or cross-linked SPF. In someembodiments, the tissue filler is administered into a dermal region ofthe subject. In some embodiments, the method is an augmentation, areconstruction, treating a disease, treating a disorder, correcting adefect or imperfection of a body part, region or area. In someembodiments, the method is a facial augmentation, a facialreconstruction, treating a facial disease, treating a facial disorder,treating a facial defect, or treating a facial imperfection.

In some embodiments of the methods described herein, the tissue fillerresists biodegradation, bioerosion, bioabsorption, and/or bioresorption,for at least about 3 days, about 7 days, about 14 days, about 21 days,about 28 days, about 1 month, about 2 months, about 3 months, about 4months, about 5 months, or about 6 months. In some embodiments of themethods described herein, administration of the tissue filler to thesubject results in a reduced inflammatory response compared to theinflammatory response induced by a control tissue filler comprising apolysaccharide and lidocaine, wherein the control tissue filler does notinclude silk protein fragments (SPF).

In some embodiments of the methods described herein, administration ofthe tissue filler to the subject results in increased collagenproduction compared to the collagen production induced by a controltissue filler comprising a polysaccharide and lidocaine, wherein thecontrol tissue filler does not include silk protein fragments (SPF).

BRIEF DESCRIPTION OF THE DRAWINGS

The presently disclosed embodiments will be further explained withreference to the attached drawings. The drawings shown are notnecessarily to scale, with emphasis instead generally being placed uponillustrating the principles of the presently disclosed embodiments.

FIG. 1 is a flow chart showing various embodiments for producing puresilk fibroin-based protein fragments (SPFs) of the present disclosure.

FIG. 2 is a flow chart showing various parameters that can be modifiedduring the process of producing SPFs of the present disclosure duringthe extraction and the dissolution steps.

FIG. 3 is a table summarizing the LiBr and Sodium Carbonate (Na₂CO₃)concentration in silk protein solutions of the present disclosure.

FIG. 4 is a table summarizing the LiBr and Na₂CO₃ concentration in silkprotein solutions of the present disclosure.

FIG. 5 is a table summarizing the Molecular Weights of silk proteinsolutions of the present disclosure.

FIGS. 6 and 7 are graphs representing the effect of extraction volume on% mass loss.

FIG. 8 is a table summarizing the Molecular Weights of silk dissolvedfrom different concentrations of LiBr and from different extraction anddissolution sizes.

FIG. 9 is a graph summarizing the effect of Extraction Time on MolecularWeight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. LiBr and 100° C. Oven Dissolution (Oven/DissolutionTime was varied).

FIG. 10 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, boiling LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 11 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 60° C. LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 12 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 80° C. LiBr and 80° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 13 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 80° C. LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 14 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 100° C. LiBr and 60° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 15 is a graph summarizing the effect of Extraction Time onMolecular Weight of silk processed under the conditions of 100° C.Extraction Temperature, 140° C. LiBr and 140° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 16 is a graph summarizing the effect of Extraction Temperature onMolecular Weight of silk processed under the conditions of 60 minuteExtraction Time, 100° C. LiBr and 100° C. Oven Dissolution(Oven/Dissolution Time was varied).

FIG. 17 is a graph summarizing the effect of LiBr Temperature onMolecular Weight of silk processed under the conditions of 60 minuteExtraction Time, 100° C. Extraction Temperature and 60° C. OvenDissolution (Oven/Dissolution Time was varied).

FIG. 18 is a graph summarizing the effect of LiBr Temperature onMolecular Weight of silk processed under the conditions of 30 minuteExtraction Time, 100° C. Extraction Temperature and 60° C. OvenDissolution (Oven/Dissolution Time was varied).

FIG. 19 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 30 minute Extraction Time, and 100°C. Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 20 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60 minute Extraction Time, and 100°C. Lithium Bromide. (Oven/Dissolution Time was varied).

FIG. 21 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60 minute Extraction Time, and 140°C. Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 22 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 30 minute Extraction Time, and 140°C. Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 23 is a graph summarizing the effect of Oven/DissolutionTemperature on Molecular Weight of silk processed under the conditionsof 100° C. Extraction Temperature, 60 minute Extraction Time, and 80° C.Lithium Bromide (Oven/Dissolution Time was varied).

FIG. 24 is a graph summarizing the Molecular Weights of silk processedunder varying conditions including Extraction Time, ExtractionTemperature, Lithium Bromide (LiBr) Temperature, Oven Temperature forDissolution, Oven Time for Dissolution.

FIG. 25 is a graph summarizing the Molecular Weights of silk processedunder conditions in which Oven/Dissolution Temperature is equal to LiBrTemperature.

FIG. 26 is a picture of silk/HA formulations in water orphosphate-buffered saline (PBS) at various concentrations, whichdemonstrate that silk/HA formulations result in homogenous, opaquesolutions. The first unmarked vial is a control vial (22 mg/mL HA inwater).

FIG. 27 is a picture of aqueous silk/HA formulations deposited insyringes, which demonstrate that silk/HA formulations result inhomogenous, opaque solutions. The control is a solution of 22 mg/mL HAin water.

FIG. 28 is a chart depicting the degradation profile of silk-HA and HAhydrogels.

FIG. 29 is a picture of an intradermal area in a guinea pig injectedwith a control dermal filler (commercially available HA filler includinglidocaine); the increased degree of inflammation is reflected by theextent of granulomatous areas. The commercially available filler isnoted as blue/gray material. Granulomatous inflammation associated withthe material can be observed at 7 days.

FIG. 30 is a picture of an intradermal area in a guinea pig injectedwith a control dermal filler (commercially available HA filler includinglidocaine); the commercially available product is noted as blue/graymaterial. At 30 days, inflammation with fibrosis can be observed.

FIG. 31 is a picture of an intradermal area in a guinea pig injectedwith a silk-HA dermal filler of the invention (24 mg/ml HA, 9.6 mg/mlsilk, BDDE cross linked); the reduced granulomatous areas as compared tothe control injection indicates negligible acute inflammatory response,and a better biodegradability of the silk-HA filler compared to thecontrol. There is very little inflammation at 7 days. The inflammationis focal and at times hard to find. No implant material is noted.

FIG. 32 is a picture of an intradermal area in a guinea pig injectedwith a silk-HA dermal filler of the invention (24 mg/ml HA, 9.6 mg/mlsilk, BDDE cross linked); at 30 days the inflammation is extremelydifficult to find and minimal. No implant material is noted.

FIG. 33 is a picture of an intradermal area in a guinea pig injectedwith a silk-HA dermal filler of the invention (24 mg/ml HA, 0.48 mg/mlsilk, BDDE cross linked); the filler results in focal mild inflammationin the 7 days. The inflammation is chronic. This inflammation requiredclose evaluation to identify since it was focal and minimal. No implantmaterial is observed.

FIG. 34 is a picture of an intradermal area in a guinea pig injectedwith a silk-HA dermal filler of the invention (24 mg/ml HA, 0.48 mg/mlsilk, BDDE cross linked); the 30-day image demonstrates even lessinflammation. It was even more difficult to identify as compared to the7 day implants. No implant material is observed.

FIG. 35 is a chart depicting turbidity measurement of a silk-HAhydrogel. Black curve (a): standard transmittance; Red curve (b):transmittance plus forward scatter.

FIG. 36 is a chart depicting turbidity measurement of HA hydrogelwithout silk. Black curve (a): standard transmittance; Red curve (b):transmittance plus forward scatter.

FIG. 37 is a representative histology picture of an intradermal area ina guinea pig injected with a control dermal filler.

FIG. 38 is a representative histology picture of an intradermal area ina guinea pig injected with an HA dermal filler of the invention (24mg/ml HA, PEGDE cross linked, Sample C4—Table 25).

FIG. 39 is a representative histology picture of an intradermal area ina guinea pig injected with a silk-HA dermal filler of the invention(22.8 mg/ml HA, 1.2 mg/ml silk, PEGDE cross linked, Sample L—Table 25).

FIG. 40 is a representative histology picture of an intradermal area ina guinea pig injected with a silk-HA dermal filler of the invention(23.76 mg/ml HA, 0.24 mg/ml silk, PEGDE cross linked, Sample M—Table25).

FIG. 41 is a representative histology picture of an intradermal area ina guinea pig injected with a silk-HA dermal filler of the invention(22.8 mg/ml HA, 1.2 mg/ml silk, PEGDE cross linked, Sample N—Table 25).

FIG. 42 is a representative histology picture of an intradermal area ina guinea pig injected with a silk-HA dermal filler of the invention(22.8 mg/ml HA, 1.2 mg/ml silk, PEGDE cross linked, Sample O—Table 25).

FIG. 43 is a graph showing 7-day post-implantation histology results forgel degradation (Table 25 formulations—BDDE cross-linked formulationsare mostly degraded; scoring: 0—normal; 1—minimal; 2—mild; 3—moderate;and 4—severe).

FIG. 44 is a graph showing 7-day post-implantation histology results forgel migration (Table 25 formulations; scoring: 0—normal; 1—minimal;2—mild; 3—moderate; and 4—severe).

FIG. 45 is a graph showing 7-day post-implantation histology results forinflammation (Table 25 formulations—no tissue necrosis was observed, noblood clotting was observed, and minimal collagen deposition wasobserved on the control formulation and some of the test formulations;scoring: 0—normal; 1—minimal; 2—mild; 3—moderate; and 4—severe).

FIG. 46 is a graph showing 7-day post-implantation histology results formacrophages (Table 25 formulations; scoring: 0—normal; 1—minimal;2—mild; 3—moderate; and 4—severe).

FIGS. 47A and 47B show the G′ of hydrogels with various silkconcentrations before and after dialysis. FIG. 47A: mixed HAcross-linked at 100 gm/ml, and FIG. 47B: single MW HA cross-linked at 25mg/ml.

FIGS. 48A and 48B show the swelling ratio of hydrogel with various silkconcentrations during dialysis. FIG. 48A: mixed HA cross-linked at 100mg/ml, and FIG. 48B: single MW HA cross-linked at 25 mg/ml.

FIGS. 49A and 49B show the calibration curves for medium and lowmolecular weight silk solutions, respectively.

FIGS. 50A and 50B show the absorbance spectra of diluted silk-HA gelswith unknown silk concentration; the theoretical silk concentration(mg/ml) is shown for each silk-HA gel sample in Table 26.

FIG. 51 shows turbidity measurement of HA hydrogel without silk (red;higher transmittance across the entire wavelength interval) and with 3mg/ml silk (blue; lower transmittance across the entire wavelengthinterval); a higher % transmittance indicates a less turbid sample, withless optical opacity.

While the above-identified drawings set forth presently disclosedembodiments, other embodiments are also contemplated, as noted in thediscussion. This disclosure presents illustrative embodiments by way ofrepresentation and not limitation. Numerous other modifications andembodiments can be devised by those skilled in the art which fall withinthe scope and spirit of the principles of the presently disclosedembodiments.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are tissue fillers that include silk protein fragments(SPF). In some embodiments, the tissue fillers are prepared fromcompositions described herein that may include SPF and hyaluronic acid(HA). In some embodiments, the tissue fillers described herein may bedermal fillers.

In some embodiments, the dermal fillers are made by a process describedherein by using HA having a MW of between about 5 kDa and about 5 MDa,between about 100 kDa and about 4 MDa, or between about 500 kDa andabout 3 MDa. In some embodiments, the dermal fillers are made by aprocess described herein by using HA having a MW of about 50 kDa, about100 kDa, about 150 kDa, about 200 kDa, about 250 kDa, about 300 kDa,about 350 kDa, about 400 kDa, about 450 kDa, about 500 kDa, about 550kDa, about 600 kDa, about 650 kDa, about 700 kDa, about 750 kDa, about800 kDa, about 850 kDa, about 900 kDa, about 950 kDa, about 1000 kDa,about 1050 kDa, about 1100 kDa, about 1150 kDa, about 1200 kDa, about1250 kDa, about 1300 kDa, about 1350 kDa, about 1400 kDa, about 1450kDa, about 1500 kDa, about 1550 kDa, about 1600 kDa, about 1650 kDa,about 1700 kDa, about 1750 kDa, about 1800 kDa, about 1850 kDa, about1900 kDa, about 1950 kDa, about 2000 kDa, about 2050 kDa, about 2100kDa, about 2150 kDa, about 2200 kDa, about 2250 kDa, about 2300 kDa,about 2350 kDa, about 2400 kDa, about 2450 kDa, about 2500 kDa, about2550 kDa, about 2600 kDa, about 2650 kDa, about 2700 kDa, about 2750kDa, about 2800 kDa, about 2850 kDa, about 2900 kDa, about 2950 kDa,about 3000 kDa, about 3050 kDa, about 3100 kDa, about 3150 kDa, about3200 kDa, about 3250 kDa, about 3300 kDa, about 3350 kDa, about 3400kDa, about 3450 kDa, about 3500 kDa, about 3550 kDa, about 3600 kDa,about 3650 kDa, about 3700 kDa, about 3750 kDa, about 3800 kDa, about3850 kDa, about 3900 kDa, about 3950 kDa, or about 4000 kDa. Any of theabove MW of HA can be mixed with any other of the above MW of HA, in anypossible proportion. In some embodiments, a dermal filler is made bymixing a high MW HA can be mixed with a low MW HA, where the high MW HAis in a proportion of about 0.01%, or about 0.1%, or about 0.2%, orabout 0.3%, or about 0.4%, or about 0.5%, or about 0.6%, or about 0.7%,or about 0.8%, or about 0.9%, or about 1%, or about 2%, or about 3%, orabout 4%, or about 5%, or about 6%, or about 7%, or about 8%, or about9%, or about 10%, or about 11%, or about 12%, or about 13%, or about14%, or about 15%, or about 16%, or about 17%, or about 18%, or about19%, or about 20%, or about 21%, or about 22%, or about 23%, or about24%, or about 25%, or about 26%, or about 27%, or about 28%, or about29%, or about 30%, or about 31%, or about 32%, or about 33%, or about34%, or about 35%, or about 36%, or about 37%, or about 38%, or about39%, or about 40%, or about 41%, or about 42%, or about 43%, or about44%, or about 45%, or about 46%, or about 47%, or about 48%, or about49%, or about 50%, or about 51%, or about 52%, or about 53%, or about54%, or about 55%, or about 56%, or about 57%, or about 58%, or about59%, or about 60%, or about 61%, or about 62%, or about 63%, or about64%, or about 65%, or about 66%, or about 67%, or about 68%, or about69%, or about 70%, or about 71%, or about 72%, or about 73%, or about74%, or about 75%, or about 76%, or about 77%, or about 78%, or about79%, or about 80%, or about 81%, or about 82%, or about 83%, or about84%, or about 85%, or about 86%, or about 87%, or about 88%, or about89%, or about 90%, or about 91%, or about 92%, or about 93%, or about94%, or about 95%, or about 96%, or about 97%, or about 98%, or about99%, or about 99.5%, or about 99.9%.

In some embodiments, the dermal fillers are made by a process describedherein by using silk SPF having a MW between about 5 kDa and about 35kDa. In some embodiments, the dermal fillers are made by a processdescribed herein by using silk SPF having a MW of about 5 kDa, or about6 kDa, or about 7 kDa, or about 8 kDa, or about 9 kDa, or about 10 kDa,or about 11 kDa, or about 12 kDa, or about 13 kDa, or about 14 kDa, orabout 15 kDa, or about 16 kDa, or about 17 kDa, or about 19 kDa, orabout 19 kDa, or about 20 kDa, or about 21 kDa, or about 22 kDa, orabout 23 kDa, or about 24 kDa, or about 25 kDa, or about 26 kDa, orabout 27 kDa, or about 28 kDa, or about 29 kDa, or about 30 kDa.

In some embodiments, the dermal fillers are made by a process describedherein by using an initial concentration of HA of about 80 mg/ml, orabout 81 mg/ml, or about 82 mg, ml, or about 83 mg/ml, or about 84mg/ml, or about 85 mg/ml, or about 86 mg/ml, or about 87 mg/ml, or about88 mg/ml, or about 89 mg/ml, or about 90 mg/ml, or about 91 mg/ml, orabout 92 mg/ml, or about 93 mg/ml, or about 94 mg/ml, or about 95 mg/ml,or about 96 mg/ml, or about 97 mg/ml, or about 98 mg/ml, or about 99mg/ml, or about 100 mg/ml, or about 101 mg/ml, or about 102 mg/ml, orabout 103 mg/ml, or about 104 mg/ml, or about 105 mg/ml, or about 106mg/ml, or about 107 mg/ml, or about 108 mg/ml, or about 109 mg/ml, orabout 110 mg/ml, or about 111 mg/ml, or about 112 mg/ml, or about 113mg/ml, or about 114 mg/ml, or about 115 mg/ml, or about 116 mg/ml, orabout 117 mg/ml, or about 118 mg/ml, or about 119 mg/ml, or about 120mg/ml, or higher.

In some embodiments, the dermal fillers described herein have a silk SPFconcentration of about 0.1%, or about 0.2%, or about 0.3%, or about0.4%, or about 0.5%, or about 0.6%, or about 0.7%, or about 0.8%, orabout 0.9%, or about 1%, or about 1.1%, or about 1.2%, or about 1.3%, orabout 1.4%, or about 1.5%, or about 1.6%, or about 1.7%, or about 1.8%,or about 1.9%, or about 2%, or about 2.1%, or about 2.2%, or about 2.3%,or about 2.4%, or about 2.5%, or about 2.6%, or about 2.7%, or about2.8%, or about 2.9%, or about 3%, or about 3.1%, or about 3.2%, or about3.3%, or about 3.4%, or about 3.5%, or about 3.6%, or about 3.7%, orabout 3.8%, or about 3.9%, or about 4%, or about 4.1%, or about 4.2%, orabout 4.3%, or about 4.4%, or about 4.5%, or about 4.6%, or about 4.7%,or about 4.8%, or about 4.9%, or about 5% of total HA and silk SPF.

In some embodiments, the dermal fillers are made by a process describedherein by using a PEGDE cross-linker having a Mn of about 100, about200, about 300, about 400, about 500, about 600, about 700, about 800,about 900, about 1000, about 1100, or about 1200.

In some embodiments, the dermal fillers are made by a process describedherein by using reaction conditions including a cross-linking step atabout 35° C., about 36° C., about 37° C., about 38° C., about 39° C.,about 40° C., about 41° C., about 42° C., about 43° C., about 44° C.,about 45° C., about 46° C., about 47° C., about 48° C., about 49° C.,about 50° C., about 51° C., about 52° C., about 53° C., about 54° C., orabout 55° C. In some embodiments, the dermal fillers are made by aprocess described herein by using reaction conditions including across-linking step of about 15 minutes, about 16 minutes, about 17minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29minutes, about 30 minutes, about 31 minutes, about 32 minutes, about 33minutes, about 34 minutes, about 35 minutes, about 36 minutes, about 37minutes, about 38 minutes, about 39 minutes, about 40 minutes, about 41minutes, about 42 minutes, about 43 minutes, about 44 minutes, about 45minutes, about 46 minutes, about 47 minutes, about 48 minutes, about 49minutes, about 50 minutes, about 51 minutes, about 52 minutes, about 53minutes, about 54 minutes, about 55 minutes, about 56 minutes, about 57minutes, about 58 minutes, about 59 minutes, about 60 minutes, about 61minutes, about 62 minutes, about 63 minutes, about 64 minutes, or about65 minutes.

In some embodiments, the dermal fillers include free HA, for exampleun-cross-linked HA. In some embodiments, the dermal fillers includeabout 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, or about 0.5%,or about 0.6%, or about 0.7%, or about 0.8%, or about 0.9%, or about 1%,or about 1.1%, or about 1.2%, or about 1.3%, or about 1.4%, or about1.5%, or about 1.6%, or about 1.7%, or about 1.8%, or about 1.9%, orabout 2%, or about 2.1%, or about 2.2%, or about 2.3%, or about 2.4%, orabout 2.5%, or about 2.6%, or about 2.7%, or about 2.8%, or about 2.9%,or about 3%, or about 3.1%, or about 3.2%, or about 3.3%, or about 3.4%,or about 3.5%, or about 3.6%, or about 3.7%, or about 3.8%, or about3.9%, or about 4%, or about 4.1%, or about 4.2%, or about 4.3%, or about4.4%, or about 4.5%, or about 4.6%, or about 4.7%, or about 4.8%, orabout 4.9%, or about 5%, about 5.1%, or about 5.2%, or about 5.3%, orabout 5.4%, or about 5.5%, or about 5.6%, or about 5.7%, or about 5.8%,or about 5.9%, or about 6%, or about 6.1%, or about 6.2%, or about 6.3%,or about 6.4%, or about 6.5%, or about 6.6%, or about 6.7%, or about6.8%, or about 6.9%, or about 7%, or about 7.1%, or about 7.2%, or about7.3%, or about 7.4%, or about 7.5%, or about 7.6%, or about 7.7%, orabout 7.8%, or about 7.9%, or about 8%, or about 8.1%, or about 8.2%, orabout 8.3%, or about 8.4%, or about 8.5%, or about 8.6%, or about 8.7%,or about 8.8%, or about 8.9%, or about 9%, or about 9.1%, or about 9.2%,or about 9.3%, or about 9.4%, or about 9.5%, or about 9.6%, or about9.7%, or about 9.8%, or about 9.9%, or about 10% of total HA(cross-linked HA and un-cross-linked HA). In some embodiments, thedermal fillers do not include free HA.

In some embodiments, the dermal fillers include HA at about 10 mg/ml,about 11 mg/ml, about 12 mg/ml, about 13 mg/ml, about 14 mg/ml, about 15mg/ml, about 16 mg/ml, about 17 mg/ml, about 18 mg/ml, about 19 mg/ml,about 20 mg/ml, about 21 mg/ml, about 22 mg/ml, about 23 mg/ml, about 24mg/ml, about 25 mg/ml, about 26/mg/ml, about 27 mg/ml, about 28 mg/ml,about 29 mg/ml, or about 30 mg/ml.

In some embodiments, the dermal fillers have a MoD of about 10.0%, about10.1%, about 10.2%, about 10.3%, about 10.4%, about 10.5%, about 10.6%,about 10.7%, about 10.8%, about 10.9%, about 11.0%, about 11.1%, about11.2%, about 11.3%, about 11.4%, about 11.5%, about 11.6%, about 11.7%,about 11.8%, about 11.9%, about 12.0%, about 12.1%, about 12.2%, about12.3%, about 12.4%, about 12.5%, about 12.6%, about 12.7%, about 12.8%,about 12.9%, about 13.0%, about 13.1%, about 13.2%, about 13.3%, about13.4%, about 13.5%, about 13.6%, about 13.7%, about 13.8%, about 13.9%,about 14.0%, about 14.1%, about 14.2%, about 14.3%, about 14.4%, about14.5%, about 14.6%, about 14.7%, about 14.8%, about 14.9%, about 15.0%,about 15.1%, about 15.2%, about 15.3%, about 15.4%, about 15.5%, about15.6%, about 15.7%, about 15.8%, about 15.9%, about 16.0%, about 16.1%,about 16.2%, about 16.3%, about 16.4%, about 16.5%, about 16.6%, about16.7%, about 16.8%, about 16.9%, about 17.0%, about 17.1%, about 17.2%,about 17.3%, about 17.4%, about 17.5%, about 17.6%, about 17.7%, about17.8%, about 17.9%, about 18.0%, about 18.1%, about 18.2%, about 18.3%,about 18.4%, about 18.5%, about 18.6%, about 18.7%, about 18.8%, about18.9%, about 19.0%, about 19.1%, about 19.2%, about 19.3%, about 19.4%,about 19.5%, about 19.6%, about 19.7%, about 19.8%, about 19.9%, orabout 20.0%.

In some embodiments, the dermal fillers have an injection force of about5 N, about 6 N, about 7 N, about 8 N, about 9 N, about 10 N, about 11 N,about 12 N, about 13 N, about 14 N, about 15 N, about 16 N, about 17 N,about 18 N, about 19 N, about 20 N, about 21 N, about 22 N, about 23 N,about 24 N, or about 25 N. In some embodiments, the injection forcerelate to injection through a 30 G needle.

The tissue fillers provided herein include compositions furtherincluding one or more components such as SPF, for example cross-linkedSPF and/or non-cross-linked SPF, hyaluronic acid, for examplecross-linked HA and/or non-cross-linked HA. As used herein, cross-linkedSPF refers to SPF which is cross-linked with an identical ornon-identical SPF. Cross-linked SPF can also be referred to ashomo-cross-linked SPF. As used herein, cross-linked HA refers to HAwhich is cross-linked with an identical or non-identical HA.Cross-linked HA can also be referred to as homo-cross-linked HA. Thetissue fillers provided herein can also include SPF cross-linked to HA,and/or HA cross-linked to SPF. SPF cross-linked to HA, and/or HAcross-linked to SPF, can also be referred to as cross-linked SPF-HA, orhetero-cross-linked SPF-HA.

In some embodiments, the compositions of the invention are monophasic.In some embodiments, the compositions of the invention are biphasic, ormultiphasic. In some embodiments, the compositions of the inventioninclude a non-cross-linked polymeric phase, for example non-cross-linkedSPF, and/or non-cross-linked HA. In some embodiments, the compositionsof the invention include a cross-linked phase, for example cross-linkedSPF, and/or cross-linked HA. In some embodiments, the compositions ofthe invention include a liquid phase, for example water, and/or anaqueous solution. In some embodiments, the aqueous solution can includeSPF. In some embodiments, the aqueous phase can include HA. In someembodiments, the liquid phase may include a non-cross-linked polymersuch as non-cross-linked HA and/or non-cross-linked SPF.

In some embodiments, a composition of the invention comprises a carrierphase. As such, the disclosed compositions can be monophasic ormultiphasic compositions. As used herein, the term “carrier phase” issynonymous with “carrier” and refers to a material used to increasefluidity of a hydrogel. A carrier is advantageously aphysiologically-acceptable carrier and may include one or moreconventional excipients useful in pharmaceutical compositions. As usedherein, the term “a physiologically-acceptable carrier” refers to acarrier in accord with, or characteristic of, the normal functioning ofa living organism. As such, administration of a composition comprising ahydrogel and a carrier has substantially no long term or permanentdetrimental effect when administered to a mammal. The present tissuefillers include a carrier where a major of the volume is water orsaline. However, other useful carriers include any physiologicallytolerable material which improves upon extrudability or intrudability ofthe hydrogel through a needle or into a target host environment.Potential carriers could include but are not limited to physiologicalbuffer solutions, serum, other protein solutions, gels composed ofpolymers including proteins, glycoproteins, proteoglycans, orpolysaccharides. Any of the indicated potential carriers may be eithernaturally derived, wholly synthetic, or combinations of thereof.

In one embodiment, a composition provided herein includes one or more ofmodified SPF, cross-linked SPF, non-cross-linked SPF, modified HA,cross-linked HA, non-cross-linked HA, homo-cross-linked SPF,homo-cross-linked HA, and hetero-cross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF and non-cross-linked SPF. In some embodiments, thecompositions provided herein include cross-linked SPF andnon-cross-linked HA. In some embodiments, the compositions providedherein include cross-linked SPF and cross-linked HA. In someembodiments, the compositions provided herein include cross-linked SPFand cross-linked SPF-HA.

In some embodiments, the compositions provided herein includenon-cross-linked SPF and non-cross-linked HA. In some embodiments, thecompositions provided herein include non-cross-linked SPF andcross-linked HA. In some embodiments, the compositions provided hereininclude non-cross-linked SPF and cross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF, non-cross-linked SPF, and non-cross-linked HA. In someembodiments, the compositions provided herein include cross-linked SPF,non-cross-linked SPF, and cross-linked HA. In some embodiments, thecompositions provided herein include cross-linked SPF, non-cross-linkedSPF, and cross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF, cross-linked HA, and non-cross-linked HA. In someembodiments, the compositions provided herein include cross-linked SPF,cross-linked HA, and cross-linked SPF-HA. In some embodiments, thecompositions provided herein include cross-linked SPF, non-cross-linkedHA, and cross-linked SPF-HA.

In some embodiments, the compositions provided herein includenon-cross-linked SPF, cross-linked HA, and non-cross-linked HA. In someembodiments, the compositions provided herein include non-cross-linkedSPF, cross-linked HA, and cross-linked SPF-HA. In some embodiments, thecompositions provided herein include non-cross-linked SPF,non-cross-linked HA, and cross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF, non-cross-linked SPF, cross-linked HA, andnon-cross-linked HA. In some embodiments, the compositions providedherein include cross-linked SPF, non-cross-linked SPF, cross-linked HA,and cross-linked SPF-HA. In some embodiments, the compositions providedherein include cross-linked SPF, non-cross-linked SPF, non-cross-linkedHA, and cross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF, cross-linked HA, non-cross-linked HA, and cross-linkedSPF-HA. In some embodiments, the compositions provided herein includenon-cross-linked SPF, cross-linked HA, non-cross-linked HA, andcross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF, non-cross-linked SPF, cross-linked HA,non-cross-linked HA, and cross-linked SPF-HA.

In some embodiments, the compositions provided herein includecross-linked SPF. In some embodiments, the compositions provided hereininclude SPF and hyaluronic acids (HA). In one aspect, the SPF/HA basedcompositions described herein include HA cross-linked moieties. In someembodiments, the compositions include SPF-HA cross linked moieties. Insome embodiments, the compositions include non-cross linked HA. In someembodiments, the compositions may include non-cross linked SPF. In someembodiments, the compositions may include at least one additional agent.In some embodiments, the compositions include cross-linked SPF-SPF,SPF-HA, and or HA-HA, with variable stability, resulting in compositionsof various degrees of bio absorbability, and/or bioresorbability.

In some embodiments, the HA is cross-linked into a matrix. In someembodiments, the HA matrix encapsulates or semi-encapsulates one or moreSPF. In some embodiments, the HA is cross-linked with one or more SPF.

In some embodiments, the tissue fillers, or portions thereof, arebiocompatible, biodegradable, bioabsorbable, bioresorbable, or acombination thereof. In some embodiments, the tissue fillers providedherein include a fluid component, for example a single fluid or asolution including substantially one or more fluids. In someembodiments, the tissue fillers include water or an aqueous solution. Insome embodiments, the tissue fillers are injectable, implantable, ordeliverable under the skin by any means known in the art such as, forexample, following surgical resection of the tissue. In someembodiments, the compositions are dermal fillers. In some embodiments,the compositions are sterile.

In some embodiments, the tissue fillers described herein may includeabout 1% (w/w) SPF and about 0.3% (w/w) lidocaine.

Provided herein are methods of manufacturing compositions including silkprotein fragments (SPFs) and hyaluronic acid (HA), methods of deliveryof compositions including SPF and HA, and methods of treatment usingcompositions including SPF and HA.

Definitions

As used herein, the term “fibroin” includes silkworm fibroin, insect orspider silk protein, or recombinant silk fibroin. In an embodiment,fibroin is obtained from Bombyx mori.

As used herein, the terms “substantially sericin free” or “substantiallydevoid of sericin” refer to silk fibers in which a majority of thesericin protein has been removed, and/or SPF made from silk fibers inwhich a majority of the sericin protein has been removed. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 0.01% (w/w)and about 10.0% (w/w) sericin. In an embodiment, silk fibroin and SPFthat are substantially devoid of sericin refers to silk fibroin and SPFhaving between about 0.01% (w/w) and about 9.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 0.01% (w/w)and about 8.0% (w/w) sericin. In an embodiment, silk fibroin and SPFthat are substantially devoid of sericin refers to silk fibroin and SPFhaving between about 0.01% (w/w) and about 7.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 0.01% (w/w)and about 6.0% (w/w) sericin. In an embodiment, silk fibroin and SPFthat are substantially devoid of sericin refers to silk fibroin and SPFhaving between about 0.01% (w/w) and about 5.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 0% (w/w) andabout 4.0% (w/w) sericin. In an embodiment, silk fibroin and SPF thatare substantially devoid of sericin refers to silk fibroin and SPFhaving between about 0.05% (w/w) and about 4.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 0.1% (w/w)and about 4.0% (w/w) sericin. In an embodiment, silk fibroin and SPFthat are substantially devoid of sericin refers to silk fibroin and SPFhaving between about 0.5% (w/w) and about 4.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 1.0% (w/w)and about 4.0% (w/w) sericin. In an embodiment, silk fibroin and SPFthat are substantially devoid of sericin refers to silk fibroin and SPFhaving between about 1.5% (w/w) and about 4.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having between about 2.0% (w/w)and about 4.0% (w/w) sericin. In an embodiment, silk fibroin and SPFthat are substantially devoid of sericin refers to silk fibroin and SPFhaving between about 2.5% (w/w) and about 4.0% (w/w) sericin. In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having a sericin content betweenabout 0.01% (w/w) and about 0.1% (w/w). In an embodiment, silk fibroinand SPF that are substantially devoid of sericin refers to silk fibroinand SPF having a sericin content below about 0.1% (w/w). In anembodiment, silk fibroin and SPF that are substantially devoid ofsericin refers to silk fibroin and SPF having a sericin content belowabout 0.05% (w/w). In an embodiment, when a silk source is added to aboiling (100° C.) aqueous solution of sodium carbonate for a treatmenttime of between about 30 minutes to about 60 minutes, a degumming lossof about 26 wt. % to about 31 wt. % is obtained.

As used herein, the term “substantially homogeneous” may refer to puresilk fibroin-based protein fragments that are distributed in a normaldistribution about an identified molecular weight. As used herein, theterm “substantially homogeneous” may refer to an even distribution of anadditive, for example lidocaine, throughout a composition of the presentdisclosure.

As used herein, the term “substantially free of inorganic residuals”means that the composition exhibits residuals of 0.1% (w/w) or less. Inan embodiment, substantially free of inorganic residuals refers to acomposition that exhibits residuals of 0.05% (w/w) or less. In anembodiment, substantially free of inorganic residuals refers to acomposition that exhibits residuals of 0.01% (w/w) or less. In anembodiment, the amount of inorganic residuals is between 0 ppm(“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount ofinorganic residuals is ND to about 500 ppm. In an embodiment, the amountof inorganic residuals is ND to about 400 ppm. In an embodiment, theamount of inorganic residuals is ND to about 300 ppm. In an embodiment,the amount of inorganic residuals is ND to about 200 ppm. In anembodiment, the amount of inorganic residuals is ND to about 100 ppm. Inan embodiment, the amount of inorganic residuals is between 10 ppm and1000 ppm.

As used herein, the term “substantially free of organic residuals” meansthat the composition exhibits residuals of 0.1% (w/w) or less. In anembodiment, substantially free of organic residuals refers to acomposition that exhibits residuals of 0.05% (w/w) or less. In anembodiment, substantially free of organic residuals refers to acomposition that exhibits residuals of 0.01% (w/w) or less. In anembodiment, the amount of organic residuals is between 0 ppm(“non-detectable” or “ND”) and 1000 ppm. In an embodiment, the amount oforganic residuals is ND to about 500 ppm. In an embodiment, the amountof organic residuals is ND to about 400 ppm. In an embodiment, theamount of organic residuals is ND to about 300 ppm. In an embodiment,the amount of organic residuals is ND to about 200 ppm. In anembodiment, the amount of organic residuals is ND to about 100 ppm. Inan embodiment, the amount of organic residuals is between 10 ppm and1000 ppm.

As used herein, the term “non-cross-linked” refers to a lack ofintermolecular bonds joining individual matrix polymer molecules,macromolecules, and/or monomer chains. As such, a non-cross-linkedmatrix polymer is not linked to any other matrix polymer by anintermolecular bond.

Tissue fillers, compositions, or portions thereof, of the presentdisclosure exhibit “biocompatibility” or are “biocompatible” meaningthat the compositions are compatible with living tissue or a livingsystem by not being substantially toxic, injurious, or physiologicallyreactive and not causing immunological rejection. The term“biocompatible” encompasses the terms “bioabsorbable,” “bioresorbable,”and “biodegradable,” which are defined herein.

Tissue fillers, compositions, or portions thereof, of the presentdisclosure may be “bioabsorbable,” “bioresorbable,” and/or“biodegradable”. As used herein, the terms “bioabsorbable” refers tomaterials or substances that dissipate upon implantation within a body,independent of which mechanisms by which dissipation can occur, such asdissolution, degradation, absorption and excretion. As used herein, theterm “bioresorbable” means capable of being absorbed by the body. Asused herein, the term “biodegradable” refers to materials which candecompose under physiological conditions into byproducts. Suchphysiological conditions include, for example, hydrolysis (decompositionvia hydrolytic cleavage), enzymatic catalysis (enzymatic degradation),mechanical interactions, and the like. As used herein, the term“biodegradable” also encompasses the term “bioresorbable”, whichdescribes a material or substance that decomposes under physiologicalconditions to break down to products that undergo bioresorption into thehost-organism, namely, become metabolites of the biochemical systems ofthe host organism. As used herein, the terms “bioresorbable” and“bioresorption” encompass processes such as cell-mediated degradation,enzymatic degradation and/or hydrolytic degradation of the bioresorbablepolymer, and/or elimination of the bioresorbable polymer from livingtissue as will be appreciated by the person skilled in the art. In someembodiments, the SPF-HA compositions and materials described herein maybe biocompatible, bioresorbable, bioabsorbable, and/or biodegradable.

Where the tissue fillers described herein are biodegradable orbioresorbable, they may resist biodegradation or bioresorption for atleast about 1 day, or at least about 2 days, or at least about 3 days,or at least about 4 days, at least about 5 days, or at least about 10days, or at least about 15 days, or at least about 20 days, or at leastabout 25 days, or at least about 30 days, or at least about 35 days, orat least about 40 days, or at least about 45 days, or at least about 50days, or at least about 60 days, or at least about 70 days, or at leastabout 80 days, or at least about 90 days, or at least about 100 days, orat least about 110 days, or at least about 120 days, or at least about130 days, or at least about 140 days, or at least about 140 days, or atleast about 150 days, or at least about 160 days, or at least about 170days, or at least about 180 days, or at least about 190 days, or atleast about 200 days, or at least about 250 days, or at least about 300days, or at least about 1 year, or at least about 2 years or they mayresist biodegradation for less than about 5 days, or at most about 10days, or at most about 15 days, or at most about 20 days, or at mostabout 25 days, or at most about 30 days, or at most about 35 days, or atmost about 40 days, or at most about 45 days, or at most about 50 days,or at most about 60 days, or at most about 70 days, or at most about 80days, or at most about 90 days, or at most about 100 days, or at mostabout 110 days, or at most about 120 days, or at most about 130 days, orat most about 140 days, or at most about 140 days, or at most about 150days, or at most about 160 days, or at most about 170 days, or at mostabout 180 days, or at most about 190 days, or at most about 200 days, orat most about 250 days, or at most about 300 days, or at most about 1year, or at most about 2 years.

Where the tissue fillers described herein are bioabsorbable they mayresist bioabsorption for at least about 1 day, or at least about 2 days,or at least about 3 days, or at least about 4 days, at least about 5days, or at least about 10 days, or at least about 15 days, or at leastabout 20 days, or at least about 25 days, or at least about 30 days, orat least about 35 days, or at least about 40 days, or at least about 45days, or at least about 50 days, or at least about 60 days, or at leastabout 70 days, or at least about 80 days, or at least about 90 days, orat least about 100 days, or at least about 110 days, or at least about120 days, or at least about 130 days, or at least about 140 days, or atleast about 140 days, or at least about 150 days, or at least about 160days, or at least about 170 days, or at least about 180 days, or atleast about 190 days, or at least about 200 days, or at least about 250days, or at least about 300 days, or at least about 1 year, or at leastabout 2 years or they may resist bioabsorption for less than about 5days, or at most about 10 days, or at most about 15 days, or at mostabout 20 days, or at most about 25 days, or at most about 30 days, or atmost about 35 days, or at most about 40 days, or at most about 45 days,or at most about 50 days, or at most about 60 days, or at most about 70days, or at most about 80 days, or at most about 90 days, or at mostabout 100 days, or at most about 110 days, or at most about 120 days, orat most about 130 days, or at most about 140 days, or at most about 140days, or at most about 150 days, or at most about 160 days, or at mostabout 170 days, or at most about 180 days, or at most about 190 days, orat most about 200 days, or at most about 250 days, or at most about 300days, or at most about 1 year, or at most about 2 years.

As described herein, the degree of biodegradation, bioabsorption, andbioresorption may be modified and/or controlled by, for example, addingone or more agents to compositions described herein that retardbiodegradation, bioabsorption, and/or bioresorption. In addition, thedegree of biodegradation, bioabsorption, and bioresorption may bemodified and/or controlled by increasing or decreasing the degree ofpolymeric cross-linking present in the polymeric materials describedherein. For example, the rate of biodegradation, bioabsorption, and/orbioresorption of the compositions described here may be increased byreducing the amount of crosslinking in the polymeric materials describedherein. Alternatively, the rate of biodegradation, bioabsorption, and/orbioresorption of the tissue fillers and compositions described here maybe decreased by increasing the amount of crosslinking in the polymericmaterials described herein.

Tissue fillers and compositions of the present disclosure are“hypoallergenic” meaning that they are relatively unlikely to cause anallergic reaction. Such hypoallergenicity can be evidenced byparticipants topically applying compositions of the present disclosureon their skin for an extended period of time. In an embodiment, theextended period of time is about 3 days. In an embodiment, the extendedperiod of time is about 7 days. In an embodiment, the extended period oftime is about 14 days. In an embodiment, the extended period of time isabout 21 days. In an embodiment, the extended period of time is about 30days. In an embodiment, the extended period of time is selected from thegroup consisting of about 1 month, about 2 months, about 3 months, about4 months, about 5 months, about 6 months, about 7 months, about 8months, about 9 months, about 10 months, about 11 months, about 12months, and indefinitely.

As used herein, “low molecular weight” silk refers to silk proteinfragments having a molecular weight in a range of about 5 kDa to about20 kDa. In some embodiments, a target low molecular weight for certainsilk protein fragments may be about 11 kDa.

As used herein, “medium molecular weight” silk refers to silk proteinfragments having a molecular weight in a range of about 20 kDa to about55 kDa. In some embodiments, a target low molecular weight for certainsilk protein fragments may be about 40 kDa.

As used herein, “high molecular weight” silk refers to silk proteinfragments having a molecular weight in a range of about 55 kDa to about150 kDa. In some embodiments, a target low molecular weight for certainsilk protein fragments may be about 100 kDa to about 145 kDa.

In some embodiments, the molecular weights described herein, e.g., lowmolecular weight SPF, medium molecular weight SPF, high molecular weightSPF, may be converted to the approximate number of amino acids containedwithin the respective natural or recombinant proteins, such as naturalor recombinant silk proteins, as would be understood by a person havingordinary skill in the art. For example, the average weight of an aminoacid may be about 110 daltons, i.e., 110 g/mol. Therefore, in someembodiments, dividing the molecular weight of a linear protein by 110daltons may be used to approximate the number of amino acid residuescontained therein.

As used herein, the term “polydispersity” refers to a measure of thedistribution of molecular mass in a given polymer sample. Polydispersitymay be calculated by dividing the weight average molecular weight (Mw)by the number average molecular weight (Mn). As used herein, the term“weight average molecular weight” (Mw) generally refers to a molecularweight measurement that depends on the contributions of polymermolecules according to their sizes. The weight average molecular weightmay be defined by the formula:

${{Mw} = \frac{\Sigma N_{i}M_{i}^{2}}{\Sigma N_{i}M_{i}}},$

where M_(i) is the molecular weight of a chain and N_(i) is the numberof chains of that molecular weight. As used herein, the term “numberaverage molecular weight” (Mn) generally refers to a molecular weightmeasurement that is calculated by dividing the total weight of all thepolymer molecules in a sample with the total number of polymer moleculesin the sample. The number average molecular weight may be defined by theformula:

${{Mn} = \frac{\Sigma N_{i}M_{i}}{\Sigma N_{i}}},$

where M_(i) is the molecular weight of a chain and N_(i) is the numberof chains of that molecular weight. For example, a monodisperse polymer,where all polymer chains are equal has a polydispersity (Mw/Mn) of 1. Ingeneral, molecular weight averages may be determined by gel permeationchromatography (GPC) and size exclusion chromatography (SEC). The largerthe polydispersity index, the broader the molecular weight.

As used herein, the term “tissue filler” refers broadly a material thatmay be provided in and about soft tissue to add volume, add support, orotherwise treat a soft tissue deficiency. The term “tissue filler” alsoencompasses dermal fillers; however, the term “dermal filler” should notbe construed as imposing any limitations as to the location and type ofdelivery of such filler. Nevertheless, dermal fillers described hereinmay generally encompass the use and delivery of such dermal fillers atmultiple levels beneath the dermis. As used herein, the term “softtissue” may refer to those tissues that connect, support, or surroundother structures and organs of the body. For example, soft tissuesdescribed herein may include, without limitation, skin, dermal tissues,subdermal tissues, cutaneous tissues, subcutaneous tissues, intraduraltissue, muscles, tendons, ligaments, fibrous tissues, fat, blood vesselsand arteries, nerves, and synovial (intradermal) tissues.

As used herein, the term “epoxy derived cross-linker” refers to amolecular bridge between two moieties in the same or separate polymerchains, which is obtained by employing a cross-linking precursorincluding an epoxide group, for example 1,4-butanediol diglycidyl ether(BDDE), polyethylene glycol diglycidyl ether (PEGDE, or PEGDGE), or asilk fibroin or silk fibroin fragment polyepoxy linker. Without wishingto be bound by any particular theory, by reacting with a reactive centerin a polymer chain, including in the side chain of the polymer, theepoxide ring opens to form a secondary alcohol and a new bond (Scheme1). Reactive groups include, but are not limited to, nucleophilic groupssuch as carboxylic groups, amino groups, or hydroxyl groups.

As used herein, “auto cross-linking” refers to either a) cross-linkingbetween two strands of polymers of similar chemical nature, for examplecross-linking between two strands of hyaluronic acid, or cross-linkingbetween two strands of SPFs, or b) cross-linking between cross-linkinggroups on the same polymers strand to create a cyclic ester (lactone), acyclic amide, a cyclic construct including a cross-linking moiety, orthe like, for example cross-linking between two groups on the samestrand of hyaluronic acid, or cross-linking between two groups on thesame SPF strand.

As used herein, “zero-length cross linking,” and/or “cross-linkingincluding a bond,” and/or “cross-linking using an activating agent,”refers to cross-linking between two groups on either separate polymerstrands, or the same polymer strand, where the groups react directlywith each other, and no additional cross-linking moiety is insertedbetween them. Cross-linking between a carboxylic acid group and an amineor alcohol, where one of the groups is activated by an activating agent,for example a carbodiimide, is an example of zero-length cross-linking.

As used herein, the “Tyndall effect.” and/or “tyndalling,” is an adverseevent occurring in some patients administered with tissue fillers.Tyndall effect is characterized by the appearance of a bluediscoloration at the skin site where a tissue filler had been injected,which represents visible dermal filler composition seen through thetranslucent epidermis. The Tyndall effect can be seen whenlight-scattering particulate-matter is dispersed in anotherwise-light-transmitting medium, when the cross-section of particlesis in a specific range, usually somewhat below or near the wavelength ofvisible light. Under the Tyndall effect, longer-wavelength light (e.g.,red) is transmitted to a greater degree through the medium, whileshorter-wavelength light (e.g., blue) is reflected to a greater degreevia scattering, giving the overall impression that the medium is coloredblue.

Silk Protein Fragments

In some embodiments, the silk protein-based compositions and silkprotein fragments, or methods of producing the same, may include thosedescribed in U.S. Patent Application Publication Nos. 2015/00933340,2015/0094269, 2016/0193130, 2016/0022560, 2016/0022561, 2016/0022562,2016/0022563, and 2016/0222579, 2016/0281294, and U.S. Pat. Nos.9,187,538, 9,522,107, 9,517,191, 9,522,108, 9,511,012, and 9,545,369,the entirety of which are incorporated herein by reference.

As used herein, silk protein fragments (SPFs) refer generally to amixture, composition, or population of peptides and/or proteinsoriginating from silk. In some embodiments, SPFs are produced assubstantially pure and highly scalable SPF mixture solutions that may beused across multiple industries for a variety of applications. Thesolutions are generated from raw pure intact silk protein material andprocessed in order to remove any sericin and achieve the desired weightaverage molecular weight (MW) and polydispersity of the fragmentmixture. Select method parameters may be altered to achieve distinctfinal silk protein fragment characteristics depending upon the intendeduse. The resulting final fragment solution is pure silk proteinfragments and water with PPM to non-detectable levels of processcontaminants, levels acceptable in the pharmaceutical, medical andconsumer cosmetic markets. The concentration, size and polydispersity ofsilk protein fragments in the solution may further be altered dependingupon the desired use and performance requirements. In an embodiment, thepure silk fibroin-based protein fragments in the solution aresubstantially devoid of sericin, have an average weight averagemolecular weight ranging from about 1 kDa to about 250 kDa, and have apolydispersity ranging from about 1.5 and about 3.0. In an embodiment,the pure silk fibroin-based protein fragments in the solution aresubstantially devoid of sericin, have an average weight averagemolecular weight ranging from about 5 kDa to about 150 kDa, and have apolydispersity ranging from about 1.5 and about 3.0. In an embodiment,the pure silk fibroin-based protein fragments in the solution aresubstantially devoid of sericin, have an average weight averagemolecular weight ranging from about 6 kDa to about 17 kDa, and have apolydispersity ranging from about 1.5 and about 3.0. In an embodiment,the pure silk fibroin-based protein fragments in the solution aresubstantially devoid of sericin, have an average weight averagemolecular weight ranging from about 17 kDa to about 39 kDa, and have apolydispersity ranging from about 1.5 and about 3.0. In an embodiment,the pure silk fibroin-based protein fragments in the solution aresubstantially devoid of sericin, have an average weight averagemolecular weight ranging from about 39 kDa to about 80 kDa, and have apolydispersity ranging from about 1.5 and about 3.0. In an embodiment,the pure silk fibroin-based protein fragments in the solution aresubstantially devoid of sericin, have an average weight averagemolecular weight ranging from about 80 kDa to about 150 kDa, and have apolydispersity ranging from about 1.5 and about 3.0.

In an embodiment, the silk protein fragments described herein may beprepared in a solution or as a solid, whereby the solid is suspended ina physiological solution (e.g., water, saline, and the like) or a gel ofHA, as described herein. In some embodiments, the silk protein fragmentsdescribed herein may be prepared in liposomes or microspheres beforedepositing the same in a gel of HA.

In an embodiment, the silk solutions of the present disclosure may beused to generate the tissue filler compositions described herein. In anembodiment, the solutions may be used to generate gels that may behomogenized with HA and additional agents to prepare the tissue fillersdescribed herein. Depending on the silk solution utilized and themethods for casting the films or gels, various properties are achieved.

In some embodiments, the percent SPF content, by weight, in the tissuefillers described herein is at least 0.01%, or at least 0.1%, or atleast 0.2%, or at least 0.3%, or at least 0.4%, or at least 0.5%, or atleast 0.6%, or at least 0.7%, or at least 0.8%, or at least 0.9%, or atleast 1%, or at least 2%, or at least 3%, or at least 4%, or at least5%, or at least 6%, or at least 7%, or at least 8%, or at least 9%, orat least 10%, or at least 11%, or at least 12%, or at least 13%, or atleast 14%, or at least 15%, or at least 16%, or at least 17%, or atleast 18%, or at least 19%, or at least 20%, or at least 21%, or atleast 22%, or at least 23%, or at least 24%, or at least 25%, or atleast 26%, or at least 27%, or at least 28%, or at least 29%, or atleast 30%, or at least 31%, or at least 32%, or at least 33%, or atleast 34%, or at least 35%, or at least 36%, or at least 37%, or atleast 38%, or at least 39%, or at least 40%, or at least 41%, or atleast 42%, or at least 43%, or at least 44%, or at least 45%, or atleast 46%, or at least 47%, or at least 48%, or at least 49%, or atleast 50%, or at least 51%, or at least 52%, or at least 53%, or atleast 54%, or at least 55%, or at least 56%, or at least 57%, or atleast 58%, or at least 59%, or at least 60%, or at least 61%, or atleast 62%, or at least 63%, or at least 64%, or at least 65%, or atleast 66%, or at least 67%, or at least 68%, or at least 69%, or atleast 70%, or at least 71%, or at least 72%, or at least 73%, or atleast 74%, or at least 75%, or at least 76%, or at least 77%, or atleast 78%, or at least 79%, or at least 80%, or at least 81%, or atleast 82%, or at least 83%, or at least 84%, or at least 85%, or atleast 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%, or at least 96%, or at least 97%, or atleast 98%, or at least 99%, or at least 99.5%, or at least 99.9%.

In some embodiments, the percent SPF content, by weight, in the tissuefillers described herein is at most 0.01%, or at most 0.1%, or at most0.2%, or at most 0.3%, or at most 0.4%, or at most 0.5%, or at most0.6%, or at most 0.7%, or at most 0.8%, or at most 0.9%, or at most 1%,or at most 2%, or at most 3%, or at most 4%, or at most 5%, or at most6%, or at most 7%, or at most 8%, or at most 9%, or at most 10%, or atmost 11%, or at most 12%, or at most 13%, or at most 14%, or at most15%, or at most 16%, or at most 17%, or at most 18%, or at most 19%, orat most 20%, or at most 21%, or at most 22%, or at most 23%, or at most24%, or at most 25%, or at most 26%, or at most 27%, or at most 28%, orat most 29%, or at most 30%, or at most 31%, or at most 32%, or at most33%, or at most 34%, or at most 35%, or at most 36%, or at most 37%, orat most 38%, or at most 39%, or at most 40%, or at most 41%, or at most42%, or at most 43%, or at most 44%, or at most 45%, or at most 46%, orat most 47%, or at most 48%, or at most 49%, or at most 50%, or at most51%, or at most 52%, or at most 53%, or at most 54%, or at most 55%, orat most 56%, or at most 57%, or at most 58%, or at most 59%, or at most60%, or at most 61%, or at most 62%, or at most 63%, or at most 64%, orat most 65%, or at most 66%, or at most 67%, or at most 68%, or at most69%, or at most 70%, or at most 71%, or at most 72%, or at most 73%, orat most 74%, or at most 75%, or at most 76%, or at most 77%, or at most78%, or at most 79%, or at most 80%, or at most 81%, or at most 82%, orat most 83%, or at most 84%, or at most 85%, or at most 86%, or at most87%, or at most 88%, or at most 89%, or at most 90%, or at most 91%, orat most 92%, or at most 93%, or at most 94%, or at most 95%, or at most96%, or at most 97%, or at most 98%, or at most 990%, or at most 99.5%,or at most 99.9%.

In some embodiments, the percent SPF content, by weight, in the tissuefillers described herein is about 0.01%, or about 0.1%, or about 0.2%,or about 0.3%, or about 0.4%, or about 0.5%, or about 0.6%, or about0.7%, or about 0.8%, or about 0.9%, or about 1%, or about 2%, or about3%, or about 4%, or about 5%, or about 6%, or about 7%, or about 8%, orabout 9%, or about 10%, or about 11%, or about 12%, or about 13%, orabout 14%, or about 15%, or about 16%, or about 17%, or about 18%, orabout 19%, or about 20%, or about 21%, or about 22%, or about 23%, orabout 24%, or about 25%, or about 26%, or about 27%, or about 28%, orabout 29%, or about 30%, or about 31%, or about 32%, or about 33%, orabout 34%, or about 35%, or about 36%, or about 37%, or about 38%, orabout 39%, or about 40%, or about 41%, or about 42%, or about 43%, orabout 44%, or about 45%, or about 46%, or about 47%, or about 48%, orabout 49%, or about 50%, or about 51%, or about 52%, or about 53%, orabout 54%, or about 55%, or about 56%, or about 57%, or about 58%, orabout 59%, or about 60%, or about 61%, or about 62%, or about 63%, orabout 64%, or about 65%, or about 66%, or about 67%, or about 68%, orabout 69%, or about 70%, or about 71%, or about 72%, or about 73%, orabout 74%, or about 75%, or about 76%, or about 77%, or about 78%, orabout 79%, or about 80%, or about 81%, or about 82%, or about 83%, orabout 84%, or about 85%, or about 86%, or about 87%, or about 88%, orabout 89%, or about 90%, or about 91%, or about 92%, or about 93%, orabout 94%, or about 95%, or about 96%, or about 97%, or about 98%, orabout 99%, or about 99.5%, or about 99.9%.

In some embodiments, the percent SPF content, by weight, in the tissuefillers described herein is between about 0.01% to about 100%, or about0.01% to about 99.9%, or about 0.01% to about 75%; or between about 0.1%to about 95%, or about 1% to about 95%, or about 10% to about 95%; orbetween about 0.1% to about 1%, or about 0.1% to about 2%, or about 0.1%to about 3%, or about 0.1% to about 4%, or about 0.1% to about 5%, orabout 0.1% to about 6%, or about 0.1% to about 7%, or about 0.1% toabout 8%, or about 0.1% to about 9%, or about 0.1% to about 10%, orabout 0.1% to about 11%, or about 0.1% to about 12%, or about 0.1% toabout 13%, or about 0.1% to about 14%, or about 0.1% to about 15%, orabout 0.1% to about 16%, or about 0.1% to about 17%, or about 0.1% toabout 18%, or about 0.1% to about 19%, or about 0.1% to about 20%, orabout 0.1% to about 21%, or about 0.1% to about 22%, or about 0.1% toabout 23%, or about 0.1% to about 24%, or about 0.1% to about 25%; orbetween about 1% to about 2%, or about 1% to about 3%, or about 1% toabout 4%, or about 1% to about 5%, or about 1% to about 6%, or about 1%to about 7%, or about 1% to about 8%, or about 1% to about 9%, or about1% to about 10%, or about 1% to about 11%, or about 1% to about 12%, orabout 1% to about 13%, or about 1% to about 14%, or about 1% to about15%, or about 1% to about 16%, or about 1% to about 17%, or about 1% toabout 18%, or about 1% to about 19%, or about 1% to about 20%, or about1% to about 21%, or about 1% to about 22%, or about 1% to about 23%, orabout 1% to about 24%, or about 1% to about 25%; or between about 10% toabout 20%, or about 10% to about 25%, or about 10% to about 30%, orabout 10% to about 35%, or about 10% to about 40%, or about 10% to about45%, or about 10% to about 50%, or about 10% to about 55%, or about 10%to about 60%, or about 10% to about 65%, or about 10% to about 70%, orabout 10% to about 75%, or about 10% to about 80%, or about 10% to about85%, or about 10% to about 90%, or about 10% to about 100%.

The SPF described herein can have a variety of mechanical and physicalproperties depending on the degree of crystallinity of the SPF peptidesand/or proteins. In an embodiment, an SPF composition of the presentdisclosure is not soluble in an aqueous solution due to thecrystallinity of the protein. In an embodiment, an SPF composition ofthe present disclosure is soluble in an aqueous solution. In anembodiment, the SPFs of a composition of the present disclosure includea crystalline portion of about two-thirds and an amorphous region ofabout one-third. In an embodiment, the SPFs of a composition of thepresent disclosure include a crystalline portion of about one-half andan amorphous region of about one-half. In an embodiment, the SPFs of acomposition of the present disclosure include a 99% crystalline portionand a 1% amorphous region. In an embodiment, the SPFs of a compositionof the present disclosure include a 95% crystalline portion and a 5%amorphous region. In an embodiment, the SPFs of a composition of thepresent disclosure include a 90% crystalline portion and a 10% amorphousregion. In an embodiment, the SPFs of a composition of the presentdisclosure include a 85% crystalline portion and a 15% amorphous region.In an embodiment, the SPFs of a composition of the present disclosureinclude a 80% crystalline portion and a 20% amorphous region. In anembodiment, the SPFs of a composition of the present disclosure includea 75% crystalline portion and a 25% amorphous region. In an embodiment,the SPFs of a composition of the present disclosure include a 70%crystalline portion and a 30% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 65%crystalline portion and a 35% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 60%crystalline portion and a 40% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 50%crystalline portion and a 50% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 40%crystalline portion and a 60% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 35%crystalline portion and a 65% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 30%crystalline portion and a 70% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 25%crystalline portion and a 75% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 20%crystalline portion and a 80% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 15%crystalline portion and a 85% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 10%crystalline portion and a 90% amorphous region. In an embodiment, theSPFs of a composition of the present disclosure include a 5% crystallineportion and a 90% amorphous region. In an embodiment, the SPFs of acomposition of the present disclosure include a 1% crystalline portionand a 99% amorphous region.

In some embodiments, the physical and mechanical properties of the SPFvary with the degree of presence in the SPF composition of α-helixand/or random coil regions. In some embodiments, an SPF hydrogeldisclosed herein has a protein structure that is substantially-free ofα-helix and random coil regions. In aspects of these embodiments, ahydrogel has a protein structure including, e.g., about 5% α-helix andrandom coil regions, about 10% α-helix and random coil regions, about15% α-helix and random coil regions, about 20% α-helix and random coilregions, about 25% α-helix and random coil regions, about 30% α-helixand random coil regions, about 35% α-helix and random coil regions,about 40% α-helix and random coil regions, about 45% α-helix and randomcoil regions, or about 50% α-helix and random coil regions. In otheraspects of these embodiments, a hydrogel has a protein structureincluding, e.g., at most 5% α-helix and random coil regions, at most 10%α-helix and random coil regions, at most 15% α-helix and random coilregions, at most 20% α-helix and random coil regions, at most 25%α-helix and random coil regions, at most 30% α-helix and random coilregions, at most 35% α-helix and random coil regions, at most 40%α-helix and random coil regions, at most 45% α-helix and random coilregions, or at most 50% α-helix and random coil regions. In yet otheraspects of these embodiments, a hydrogel has a protein structureincluding, e.g., about 5% to about 10% α-helix and random coil regions,about 5% to about 15% α-helix and random coil regions, about 5% to about20% α-helix and random coil regions, about 5% to about 25% α-helix andrandom coil regions, about 5% to about 30% α-helix and random coilregions, about 5% to about 40% α-helix and random coil regions, about 5%to about 50% α-helix and random coil regions, about 10% to about 20%α-helix and random coil regions, about 10% to about 30% α-helix andrandom coil regions, about 15% to about 25% α-helix and random coilregions, about 15% to about 30% α-helix and random coil regions, orabout 15% to about 35% α-helix and random coil regions.

In some embodiments, SPF solution compositions of the present disclosurehave shelf stability, i.e., they will not slowly or spontaneously gelwhen stored in an aqueous solution and there, without apparentaggregation of fragments and/or increase in molecular weight over time,from 10 days to 3 years depending on storage conditions, percent silk,and number of shipments and shipment conditions. Additionally, pH may bealtered to extend shelf-life and/or support shipping conditions bypreventing premature folding and aggregation of the silk. In anembodiment, a SPF solution composition of the present disclosure has ashelf stability for up to 2 weeks at room temperature (RT). In anembodiment, a SPF solution composition of the present disclosure has ashelf stability for up to 4 weeks at RT. In an embodiment, a SPFsolution composition of the present disclosure has a shelf stability forup to 6 weeks at RT. In an embodiment, a SPF solution composition of thepresent disclosure has a shelf stability for up to 8 weeks at RT. In anembodiment, a SPF solution composition of the present disclosure has ashelf stability for up to 10 weeks at RT. In an embodiment, a SPFsolution composition of the present disclosure has a shelf stability forup to 12 weeks at RT. In an embodiment, a SPF solution composition ofthe present disclosure has a shelf stability ranging from about 4 weeksto about 52 weeks at RT. Table 1 below shows shelf stability testresults for embodiments of SPF compositions of the present disclosure.

TABLE 1 Shelf Stability of SPF Compositions of the Present Disclosure %Silk Temperature Time to Gelation 2 RT   4 weeks 2 4° C. >9 weeks 4 RT  4 weeks 4 4° C. >9 weeks 6 RT   2 weeks 6 4° C. >9 weeks

A known additive such as a vitamin (e.g., vitamin C) can be added to aSPF solution composition of the present disclosure to create a gel thatis stable from 10 days to 3 years at room temperature (RT). Bothexamples, a SPF composition and the same with an additive, can belyophilized for enhanced storage control ranging from 10 days to 10years depending on storage and shipment conditions. The lyophilized silkpowder can also be used as a raw ingredient in the medical, consumer,and electronic markets. Additionally, lyophilized silk powder can beresuspended in water, HFIP, or organic solution following storage tocreate silk solutions of varying concentrations, including higherconcentration solutions than those produced initially. In anotherembodiment, the silk fibroin-based protein fragments are dried using arototherm evaporator or other methods known in the art for creating adry protein form containing less than 10% water by mass.

The SPFs used in the tissue fillers and methods disclosed herein can bemanipulated and incorporated in various ways, for example in the form ofa solution, which may be combined with other materials (e.g., HA) toprepare the tissue filler compositions described herein. Following arenon-limiting examples of suitable ranges for various parameters in andfor preparation of the silk solutions of the present disclosure. Thesilk solutions of the present disclosure may include one or more, butnot necessarily all, of these parameters and may be prepared usingvarious combinations of ranges of such parameters.

In an embodiment, the percent silk in the solution is less than 30%. Inan embodiment, the percent silk in the solution is less than 25%. In anembodiment, the percent silk in the solution is less than 20%. In anembodiment, the percent silk in the solution is less than 19%. In anembodiment, the percent silk in the solution is less than 18%. In anembodiment, the percent silk in the solution is less than 17%. In anembodiment, the percent silk in the solution is less than 16%. In anembodiment, the percent silk in the solution is less than 15%. In anembodiment, the percent silk in the solution is less than 14%. In anembodiment, the percent silk in the solution is less than 13%. In anembodiment, the percent silk in the solution is less than 12%. In anembodiment, the percent silk in the solution is less than 11%. In anembodiment, the percent silk in the solution is less than 10%. In anembodiment, the percent silk in the solution is less than 9%. In anembodiment, the percent silk in the solution is less than 8%. In anembodiment, the percent silk in the solution is less than 7%. In anembodiment, the percent silk in the solution is less than 6%. In anembodiment, the percent silk in the solution is less than 5%. In anembodiment, the percent silk in the solution is less than 4%. In anembodiment, the percent silk in the solution is less than 3%. In anembodiment, the percent silk in the solution is less than 2%. In anembodiment, the percent silk in the solution is less than 1%. In anembodiment, the percent silk in the solution is less than 0.9%. In anembodiment, the percent silk in the solution is less than 0.8%. In anembodiment, the percent silk in the solution is less than 0.7%. In anembodiment, the percent silk in the solution is less than 0.6%. In anembodiment, the percent silk in the solution is less than 0.5%. In anembodiment, the percent silk in the solution is less than 0.4%. In anembodiment, the percent silk in the solution is less than 0.3%. In anembodiment, the percent silk in the solution is less than 0.2%. In anembodiment, the percent silk in the solution is less than 0.1%. In anembodiment, the percent silk in the solution is greater than 0.1%. In anembodiment, the percent silk in the solution is greater than 0.2%. In anembodiment, the percent silk in the solution is greater than 0.3%. In anembodiment, the percent silk in the solution is greater than 0.4%. In anembodiment, the percent silk in the solution is greater than 0.5%. In anembodiment, the percent silk in the solution is greater than 0.6%. In anembodiment, the percent silk in the solution is greater than 0.7%. In anembodiment, the percent silk in the solution is greater than 0.8%. In anembodiment, the percent silk in the solution is greater than 0.9%. In anembodiment, the percent silk in the solution is greater than 1%. In anembodiment, the percent silk in the solution is greater than 2%. In anembodiment, the percent silk in the solution is greater than 3%. In anembodiment, the percent silk in the solution is greater than 4%. In anembodiment, the percent silk in the solution is greater than 5%. In anembodiment, the percent silk in the solution is greater than 6%. In anembodiment, the percent silk in the solution is greater than 7%. In anembodiment, the percent silk in the solution is greater than 8%. In anembodiment, the percent silk in the solution is greater than 9%. In anembodiment, the percent silk in the solution is greater than 10%. In anembodiment, the percent silk in the solution is greater than 11%. In anembodiment, the percent silk in the solution is greater than 12%. In anembodiment, the percent silk in the solution is greater than 13%. In anembodiment, the percent silk in the solution is greater than 14%. In anembodiment, the percent silk in the solution is greater than 15%. In anembodiment, die percent silk in the solution is greater than 16%. In anembodiment, the percent silk in the solution is greater than 17%. In anembodiment, the percent silk in the solution is greater than 18%. In anembodiment, the percent silk in the solution is greater than 19%. In anembodiment, the percent silk in the solution is greater than 20%. In anembodiment, die percent silk in the solution is greater than 25%. In anembodiment, the percent silk in the solution is between 0.1% and 30%. Inan embodiment, the percent silk in the solution is between 0.1% and 25%.In an embodiment, the percent silk in the solution is between 0.1% and20%. In an embodiment, the percent silk in the solution is between 0.1%and 15%. In an embodiment, the percent silk in the solution is between0.1% and 10%. In an embodiment, the percent silk in the solution isbetween 0.1% and 9%. In an embodiment, the percent silk in the solutionis between 0.1% and 8%. In an embodiment, the percent silk in thesolution is between 0.1% and 7%. In an embodiment, the percent silk inthe solution is between 0.1% and 6.5%. In an embodiment, the percentsilk in the solution is between 0.1% and 6%. In an embodiment, thepercent silk in the solution is between 0.1% and 5.5%. In an embodiment,the percent silk in the solution is between 0.1% and 5%. In anembodiment, the percent silk in the solution is between 0.1% and 4.5%.In an embodiment, the percent silk in the solution is between 0.1% and4%. In an embodiment, the percent silk in the solution is between 0.1%and 3.5%. In an embodiment, the percent silk in the solution is between0.1% and 3%. In an embodiment, the percent silk in the solution isbetween 0.1% and 2.5%. In an embodiment, the percent silk in thesolution is between 0.1% and 2.0%. In an embodiment, the percent silk inthe solution is between 0.1% and 2.4%. In an embodiment, the percentsilk in the solution is between 0.5% and 5%. In an embodiment, thepercent silk in the solution is between 0.5% and 4.5%. In an embodiment,the percent silk in the solution is between 0.5% and 4%. In anembodiment, the percent silk in the solution is between 0.5% and 3.5%.In an embodiment, the percent silk in the solution is between 0.5% and3%. In an embodiment, the percent silk in the solution is between 0.5%and 2.5%. In an embodiment, the percent silk in the solution is between1 and 4%. In an embodiment, the percent silk in the solution is between1 and 3.5%. In an embodiment, the percent silk in the solution isbetween 1 and 3%. In an embodiment, the percent silk in the solution isbetween 1 and 2.5%. In an embodiment, the percent silk in the solutionis between 1 and 2.4%. In an embodiment, the percent silk in thesolution is between 1 and 2%. In an embodiment, the percent silk in thesolution is between 20% and 30%. In an embodiment, the percent silk inthe solution is between 0.1% and 6%. In an embodiment, the percent silkin the solution is between 6% and 10%. In an embodiment, the percentsilk in the solution is between 6% and 8%. In an embodiment, the percentsilk in the solution is between 6% and 9%. In an embodiment, the percentsilk in the solution is between 10% and 20%. In an embodiment, thepercent silk in the solution is between 11% and 19%. In an embodiment,the percent silk in the solution is between 12% and 18%. In anembodiment, the percent silk in the solution is between 13% and 17%. Inan embodiment, the percent silk in the solution is between 14% and 16%.

In an embodiment, the silk compositions described herein may be combinedwith HA to form a tissue filler composition. In an embodiment, thepercent silk in the tissue filler composition by weight is less than30%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 25%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 20%. In an embodiment,the percent silk in the tissue filler composition by weight is less than19%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 18%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 17%. In an embodiment,the percent silk in the tissue filler composition by weight is less than16%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 15%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 14%. In an embodiment,the percent silk in the tissue filler composition by weight is less than13%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 12%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 11%. In an embodiment,the percent silk in the tissue filler composition by weight is less than10%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 9%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 8%. In an embodiment,the percent silk in the tissue filler composition by weight is less than7%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 6%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 5%. In an embodiment,the percent silk in the tissue filler composition by weight is less than4%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 3%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 2%. In an embodiment,the percent silk in the tissue filler composition by weight is less than1%. In an embodiment, the percent silk in the tissue filler compositionby weight is less than 0.9%. In an embodiment, the percent silk in thetissue filler composition by weight is less than 0.8%. In an embodiment,the percent silk in the tissue filler composition by weight is less than0.7%. In an embodiment, the percent silk in the tissue fillercomposition by weight is less than 0.6%. In an embodiment, the percentsilk in the tissue filler composition by weight is less than 0.5%. In anembodiment, the percent silk in the tissue filler composition by weightis less than 0.4%. In an embodiment, the percent silk in the tissuefiller composition by weight is less than 0.3%. In an embodiment, thepercent silk in the tissue filler composition by weight is less than0.2%. In an embodiment, the percent silk in the tissue fillercomposition by weight is less than 0.1%. In an embodiment, the percentsilk in the tissue filler composition by weight is greater than 0.1%. Inan embodiment, the percent silk in the tissue filler composition byweight is greater than 0.2%. In an embodiment, the percent silk in thetissue filler composition by weight is greater than 0.3%. In anembodiment, the percent silk in the tissue filler composition by weightis greater than 0.4%. In an embodiment, the percent silk in the tissuefiller composition by weight is greater than 0.5%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than0.6%. In an embodiment, the percent silk in the tissue fillercomposition by weight is greater than 0.7%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than0.8%. In an embodiment, the percent silk in the tissue fillercomposition by weight is greater than 0.9%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than1%. In an embodiment, the percent silk in the tissue filler compositionby weight is greater than 2%. In an embodiment, the percent silk in thetissue filler composition by weight is greater than 3%. In anembodiment, the percent silk in the tissue filler composition by weightis greater than 4%. In an embodiment, the percent silk in the tissuefiller composition by weight is greater than 5%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than6%. In an embodiment, the percent silk in the tissue filler compositionby weight is greater than 7%. In an embodiment, the percent silk in thetissue filler composition by weight is greater than 8%. In anembodiment, the percent silk in the tissue filler composition by weightis greater than 9%. In an embodiment, the percent silk in the tissuefiller composition by weight is greater than 10%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than11%. In an embodiment, the percent silk in the tissue filler compositionby weight is greater than 12%. In an embodiment, the percent silk in thetissue filler composition by weight is greater than 13%. In anembodiment, the percent silk in the tissue filler composition by weightis greater than 14%. In an embodiment, the percent silk in the tissuefiller composition by weight is greater than 15%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than16%. In an embodiment, the percent silk in the tissue filler compositionby weight is greater than 17%. In an embodiment, the percent silk in thetissue filler composition by weight is greater than 18%. In anembodiment, the percent silk in the tissue filler composition by weightis greater than 19%. In an embodiment, the percent silk in the tissuefiller composition by weight is greater than 20%. In an embodiment, thepercent silk in the tissue filler composition by weight is greater than25%. In an embodiment, the percent silk in the tissue filler compositionby weight is between 0.1% and 30%. In an embodiment, the percent silk inthe tissue filler composition by weight is between 0.1% and 25%. In anembodiment, the percent silk in the tissue filler composition by weightis between 0.1% and 20%. In an embodiment, the percent silk in thetissue filler composition by weight is between 0.1% and 15%. In anembodiment, the percent silk in the tissue filler composition by weightis between 0.1% and 10%. In an embodiment, the percent silk in thetissue filler composition by weight is between 0.1% and 9%. In anembodiment, the percent silk in the tissue filler composition by weightis between 0.1% and 8%. In an embodiment, the percent silk in the tissuefiller composition by weight is between 0.1% and 7%. In an embodiment,the percent silk in the tissue filler composition by weight is between0.1% and 6.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.1% and 6%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.1%and 5.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.1% and 5%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.1%and 4.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.1% and 4%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.1%and 3.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.1% and 3%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.1%and 2.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.1% and 2.0%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.1%and 2.4%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.5% and 5%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.5%and 4.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.5% and 4%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.5%and 3.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.5% and 3%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 0.5%and 2.5%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 1 and 4%. In an embodiment, the percentsilk in the tissue filler composition by weight is between 1 and 3.5%.In an embodiment, the percent silk in the tissue filler composition byweight is between 1 and 3%. In an embodiment, the percent silk in thetissue filler composition by weight is between 1 and 2.5%. In anembodiment, the percent silk in the tissue filler composition by weightis between 1 and 2.4%. In an embodiment, the percent silk in the tissuefiller composition by weight is between 1 and 2%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 20%and 30%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 0.1% and 6%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 6%and 10%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 6% and 8%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 6%and 9%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 10% and 20%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 11%and 19%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 12% and 18%. In an embodiment, thepercent silk in the tissue filler composition by weight is between 13%and 17%. In an embodiment, the percent silk in the tissue fillercomposition by weight is between 14% and 16%.

In an embodiment, the percent sericin in the solution or tissue fillercomposition is non-detectable to 30%. In an embodiment, the percentsericin in the solution or tissue filler composition is non-detectableto 5%. In an embodiment, the percent sericin in the solution or tissuefiller composition is 1%. In an embodiment, the percent sericin in thesolution or tissue filler composition is 2%. In an embodiment, thepercent sericin in the solution or tissue filler composition is 3%. Inan embodiment, the percent sericin in the solution or tissue fillercomposition is 4%. In an embodiment, the percent sericin in the solutionor tissue filler composition is 5%. In an embodiment, the percentsericin in the solution or tissue filler composition is 10%. In anembodiment, the percent sericin in the solution or tissue fillercomposition is 30%.

In an embodiment, the stability of the LiBr-silk fragment solution is 0to 1 year. In an embodiment, the stability of the LiBr-silk fragmentsolution is 0 to 2 years. In an embodiment, the stability of theLiBr-silk fragment solution is 0 to 3 years. In an embodiment, thestability of the LiBr-silk fragment solution is 0 to 4 years. In anembodiment, the stability of the LiBr-silk fragment solution is 0 to 5years. In an embodiment, the stability of the LiBr-silk fragmentsolution is 1 to 2 years. In an embodiment, the stability of theLiBr-silk fragment solution is 1 to 3 years. In an embodiment, thestability of the LiBr-silk fragment solution is 1 to 4 years. In anembodiment, the stability of the LiBr-silk fragment solution is 1 to 5years. In an embodiment, the stability of the LiBr-silk fragmentsolution is 2 to 3 years. In an embodiment, the stability of theLiBr-silk fragment solution is 2 to 4 years. In an embodiment, thestability of the LiBr-silk fragment solution is 2 to 5 years. In anembodiment, the stability of the LiBr-silk fragment solution is 3 to 4years. In an embodiment, the stability of the LiBr-silk fragmentsolution is 3 to 5 years. In an embodiment, the stability of theLiBr-silk fragment solution is 4 to 5 years.

In an embodiment, the stability of a silk-fibroin based protein fragmentcompositions that may be included in the tissue fillers of the presentdisclosure is 10 days to 6 months. In an embodiment, the stability of asilk-fibroin based protein fragment compositions that may be included inthe tissue fillers of the present disclosure is 6 months to 12 months.In an embodiment, the stability of a silk-fibroin based protein fragmentcompositions that may be included in the tissue fillers of the presentdisclosure is 12 months to 18 months. In an embodiment, the stability ofa silk-fibroin based protein fragment compositions that may be includedin the tissue fillers of the present disclosure is 18 months to 24months. In an embodiment, the stability of a silk-fibroin based proteinfragment compositions that may be included in the tissue fillers of thepresent disclosure is 24 months to 30 months. In an embodiment, thestability of a silk-fibroin based protein fragment compositions that maybe included in the tissue fillers of the present disclosure is 30 monthsto 36 months. In an embodiment, the stability of a silk-fibroin basedprotein fragment compositions that may be included in the tissue fillersof the present disclosure is 36 months to 48 months. In an embodiment,the stability of a silk-fibroin based protein fragment compositions thatmay be included in the tissue fillers of the present disclosure is 48months to 60 months.

In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have having an average weightaverage molecular weight ranging from 1 kDa to 250 kDa. In anembodiment, silk fibroin-based protein fragments incorporated into thetissue fillers described herein have having an average weight averagemolecular weight ranging from 5 kDa to 150 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have having an average weight average molecular weightranging from 1 kDa to 6 kDa. In an embodiment, silk fibroin-basedprotein fragments incorporated into the tissue fillers described hereinhave an average weight average molecular weight ranging from 6 kDa to 17kDa. In an embodiment, silk fibroin-based protein fragments incorporatedinto the tissue fillers described herein have an average weight averagemolecular weight ranging from 17 kDa to 39 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 39 kDa to 80 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 80 kDa to 150 kDa.

In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 250 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 240 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 230 kDa.In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 220 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 210 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 200 kDa.In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 190 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 180 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 170 kDa.In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 160 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 150 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 140 kDa.In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 130 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 120 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 110 kDa.In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 100 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 90 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 80 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 70 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 60 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 50 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 40 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 1 kDa to 30 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 1 kDa to 20 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 kDa to 10 kDa.

In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 1 to 5 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 5 to 10 kDa. In an embodiment, silk fibroin-based protein fragmentsincorporated into the tissue fillers described herein have an averageweight average molecular weight ranging from 10 to 15 kDa. In anembodiment, silk fibroin-based protein fragments incorporated into thetissue fillers described herein have an average weight average molecularweight ranging from 15 to 20 kDa. In an embodiment, silk fibroin-basedprotein fragments incorporated into the tissue fillers described hereinhave an average weight average molecular weight ranging from 20 to 25kDa. In an embodiment, silk fibroin-based protein fragments incorporatedinto the tissue fillers described herein have an average weight averagemolecular weight ranging from 25 to 30 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 30 to 35 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 35 to 40 kDa. In anembodiment, silk fibroin-based protein fragments incorporated into thetissue fillers described herein have an average weight average molecularweight ranging from 40 to 45 kDa. In an embodiment, silk fibroin-basedprotein fragments incorporated into the tissue fillers described hereinhave an average weight average molecular weight ranging from 45 to 50kDa. In an embodiment, silk fibroin-based protein fragments incorporatedinto the tissue fillers described herein have an average weight averagemolecular weight ranging from 50 to 55 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 55 to 60 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 60 to 65 kDa. In anembodiment, silk fibroin-based protein fragments incorporated into thetissue fillers described herein have an average weight average molecularweight ranging from 65 to 70 kDa. In an embodiment, silk fibroin-basedprotein fragments incorporated into the tissue fillers described hereinhave an average weight average molecular weight ranging from 70 to 75kDa. In an embodiment, silk fibroin-based protein fragments incorporatedinto the tissue fillers described herein have an average weight averagemolecular weight ranging from 75 to 80 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 80 to 85 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 85 to 90 kDa. In anembodiment, silk fibroin-based protein fragments incorporated into thetissue fillers described herein have an average weight average molecularweight ranging from 90 to 95 kDa. In an embodiment, silk fibroin-basedprotein fragments incorporated into the tissue fillers described hereinhave an average weight average molecular weight ranging from 95 to 100kDa. In an embodiment, silk fibroin-based protein fragments incorporatedinto the tissue fillers described herein have an average weight averagemolecular weight ranging from 100 to 105 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 105 to 110 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 110 to 115 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 115 to 120 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 120 to 125 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 125 to 130 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 130 to 135 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 135 to 140 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 140 to 145 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 145 to 150 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 150 to 155 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 155 to 160 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 160 to 165 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 165 to 170 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 170 to 175 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 175 to 180 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 180 to 185 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 185 to 190 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 190 to 195 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 195 to 200 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 200 to 205 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have having an average weightaverage molecular weight ranging from 205 to 210 kDa. In an embodiment,silk fibroin-based protein fragments incorporated into the tissuefillers described herein have an average weight average molecular weightranging from 210 to 215 kDa. In an embodiment, silk fibroin-basedprotein fragments incorporated into the tissue fillers described hereinhave an average weight average molecular weight ranging from 215 to 220kDa. In an embodiment, silk fibroin-based protein fragments incorporatedinto the tissue fillers described herein have an average weight averagemolecular weight ranging from 220 to 225 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 225 to 230 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 230 to 235 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 235 to 240 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 240 to 245 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 245 to 250 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 250 to 255 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 255 to 260 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 260 to 265 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 265 to 270 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 270 to 275 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 275 to 280 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 280 to 285 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 285 to 290 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 290 to 295 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 295 to 300 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 300 to 305 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 305 to 310 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 310 to 315 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 315 to 320 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 320 to 325 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 325 to 330 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 330 to 335 kDa. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have anaverage weight average molecular weight ranging from 35 to 340 kDa. Inan embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have an average weight averagemolecular weight ranging from 340 to 345 kDa. In an embodiment, silkfibroin-based protein fragments incorporated into the tissue fillersdescribed herein have an average weight average molecular weight rangingfrom 345 to 350 kDa.

In an embodiment, the tissue fillers described herein may include silkprotein comprising one or more of low molecular weight silk, mediummolecular weight silk, and high molecular weight silk.

In an embodiment, the tissue fillers described herein may include silkprotein comprising one or more of low molecular weight silk, mediummolecular weight silk, and high molecular weight silk. In an embodiment,the tissue fillers described herein may include silk protein comprisinglow molecular weight silk and medium molecular weight silk. In anembodiment, the tissue fillers described herein may include silk proteincomprising low molecular weight silk and high molecular weight silk. Inan embodiment, the tissue fillers described herein may include silkprotein comprising medium molecular weight silk and high molecularweight silk. In an embodiment, the tissue fillers described herein mayinclude silk protein comprising low molecular weight silk, mediummolecular weight silk, and high molecular weight silk.

In an embodiment, the tissue fillers described herein may include silkprotein comprising low molecular weight silk and medium molecular weightsilk. In some embodiments, the w/w ratio between low molecular weightsilk and medium molecular weight silk is between about 99:1 to about1:99, between about 95:5 to about 5:95, between about 90:10 to about10:90, between about 75:25 to about 25:75, between about 65:35 to about35:65, or between about 55:45 to about 45:55. In some embodiments, thew/w ratio between low molecular weight silk and medium molecular weightsilk is between about 99:1 to about 55:45, between about 95:5 to about45:55, between about 90:10 to about 35:65, between about 75:25 to about15:85, between about 65:35 to about 10:90, or between about 55:45 toabout 1:99. In an embodiment, the w/w ratio between low molecular weightsilk and medium molecular weight silk is about 99:1, about 98:2, about97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about91:9, about 90:10, about 89:11, about 88:12, about 87:13, about 86:14,about 85:15, about 84:16, about 83:17, about 82:18, about 81:19, about80:20, about 79:21, about 78:22, about 77:23, about 76:24, about 75:25,about 74:26, about 73:27, about 72:28, about 71:29, about 70:30, about69:31, about 68:32, about 67:33, about 66:34, about 65:35, about 64:36,about 63:37, about 62:38, about 61:39, about 60:40, about 59:41, about58:42, about 57:43, about 56:44, about 55:45, about 54:46, about 53:47,about 52:48, about 51:49, about 50:50, about 49:51, about 48:52, about47:53, about 46:54, about 45:55, about 44:56, about 43:57, about 42:58,about 41:59, about 40:60, about 39:61, about 38:62, about 37:63, about36:64, about 35:65, about 34:66, about 33:67, about 32:68, about 31:69,about 30:70, about 29:71, about 28:72, about 27:73, about 26:74, about25:75, about 24:76, about 23:77, about 22:78, about 21:79, about 20:80,about 19:81, about 18:82, about 17:83, about 16:84, about 15:85, about14:86, about 13:87, about 12:88, about 11:89, about 10:90, about 9:91,about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97,about 2:98, or about 1:99. In an embodiment, the w/w ratio between lowmolecular weight silk and medium molecular weight silk is about 9:1,about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about2:1, or about 1:1. In an embodiment, the w/w ratio between low molecularweight silk and medium molecular weight silk is about 1:9, about 1:8,about 1:7, about 1:6, about 1:5, about 1:4, about 1:3, about 1:2, orabout 1:1.

In an embodiment, the tissue fillers described herein may include silkprotein comprising low molecular weight silk and high molecular weightsilk. In some embodiments, the w/w ratio between low molecular weightsilk and high molecular weight silk is between about 99:1 to about 1:99,between about 95:5 to about 5:95, between about 90:10 to about 10:90,between about 75:25 to about 25:75, between about 65:35 to about 35:65,or between about 55:45 to about 45:55. In some embodiments, the w/wratio between low molecular weight silk and high molecular weight silkis between about 99:1 to about 55:45, between about 95:5 to about 45:55,between about 90:10 to about 35:65, between about 75:25 to about 15:85,between about 65:35 to about 10:90, or between about 55:45 to about1:99. In an embodiment, the w/w ratio between low molecular weight silkand high molecular weight silk is about 99:1, about 98:2, about 97:3,about 96:4, about 95:5, about 94:6, about 93:7, about 92:8, about 91:9,about 90:10, about 89:11, about 88:12, about 87:13, about 86:14, about85:15, about 84:16, about 83:17, about 82:18, about 81:19, about 80:20,about 79:21, about 78:22, about 77:23, about 76:24, about 75:25, about74:26, about 73:27, about 72:28, about 71:29, about 70:30, about 69:31,about 68:32, about 67:33, about 66:34, about 65:35, about 64:36, about63:37, about 62:38, about 61:39, about 60:40, about 59:41, about 58:42,about 57:43, about 56:44, about 55:45, about 54:46, about 53:47, about52:48, about 51:49, about 50:50, about 49:51, about 48:52, about 47:53,about 46:54, about 45:55, about 44:56, about 43:57, about 42:58, about41:59, about 40:60, about 39:61, about 38:62, about 37:63, about 36:64,about 35:65, about 34:66, about 33:67, about 32:68, about 31:69, about30:70, about 29:71, about 28:72, about 27:73, about 26:74, about 25:75,about 24:76, about 23:77, about 22:78, about 21:79, about 20:80, about19:81, about 18:82, about 17:83, about 16:84, about 15:85, about 14:86,about 13:87, about 12:88, about 11:89, about 10:90, about 9:91, about8:92, about 7:93, about 6:94, about 5:95, about 4:96, about 3:97, about2:98, or about 1:99.

In an embodiment, the tissue fillers described herein may include silkprotein comprising medium molecular weight silk and high molecularweight silk. In some embodiments, the w/w ratio between medium molecularweight silk and high molecular weight silk is between about 99:1 toabout 1:99, between about 95:5 to about 5:95, between about 90:10 toabout 10:90, between about 75:25 to about 25:75, between about 65:35 toabout 35:65, or between about 55:45 to about 45:55. In some embodiments,the w/w ratio between medium molecular weight silk and high molecularweight silk is between about 99:1 to about 55:45, between about 95:5 toabout 45:55, between about 90:10 to about 35:65, between about 75:25 toabout 15:85, between about 65:35 to about 10:90, or between about 55:45to about 1:99. In an embodiment, the w/w ratio between medium molecularweight silk and high molecular weight silk is about 99:1, about 98:2,about 97:3, about 96:4, about 95:5, about 94:6, about 93:7, about 92:8,about 91:9, about 90:10, about 89:11, about 88:12, about 87:13, about86:14, about 85:15, about 84:16, about 83:17, about 82:18, about 81:19,about 80:20, about 79:21, about 78:22, about 77:23, about 76:24, about75:25, about 74:26, about 73:27, about 72:28, about 71:29, about 70:30,about 69:31, about 68:32, about 67:33, about 66:34, about 65:35, about64:36, about 63:37, about 62:38, about 61:39, about 60:40, about 59:41,about 58:42, about 57:43, about 56:44, about 55:45, about 54:46, about53:47, about 52:48, about 51:49, about 50:50, about 49:51, about 48:52,about 47:53, about 46:54, about 45:55, about 44:56, about 43:57, about42:58, about 41:59, about 40:60, about 39:61, about 38:62, about 37:63,about 36:64, about 35:65, about 34:66, about 33:67, about 32:68, about31:69, about 30:70, about 29:71, about 28:72, about 27:73, about 26:74,about 25:75, about 24:76, about 23:77, about 22:78, about 21:79, about20:80, about 19:81, about 18:82, about 17:83, about 16:84, about 15:85,about 14:86, about 13:87, about 12:88, about 11:89, about 10:90, about9:91, about 8:92, about 7:93, about 6:94, about 5:95, about 4:96, about3:97, about 2:98, or about 1:99.

In an embodiment, the tissue fillers described herein may include silkprotein comprising low molecular weight silk, medium molecular weightsilk, and high molecular weight silk. In an embodiment, the w/w ratiobetween low molecular weight silk, medium molecular weight silk, andhigh molecular weight silk is about 1:1:8, 1:2:7, 1:3:6, 1:4:5, 1:5:4,1:6:3, 1:7:2, 1:8:1, 2:1:7, 2:2:6, 2:3:5, 2:4:4, 2:5:3, 2:6:2, 2:7:1,3:1:6, 3:2:5, 3:3:4, 3:4:3, 3:5:2, 3:6:1, 4:1:5, 4:2:4, 4:3:3, 4:4:2,4:5:1, 5:1:4, 5:2:3, 5:3:2, 5:4:1, 6:1:3, 6:2:2, 6:3:1, 7:1:2, 7:2:1, or8:1:1. In an embodiment, the w/w ratio between low molecular weightsilk, medium molecular weight silk, and high molecular weight silk isabout 3:0.1:0.9, 3:0.2:0.8, 3:0.3:0.7, 3:0.4:0.6, 3:0.5:0.5, 3:0.6:0.4,3:0.7:0.3, 3:0.8:0.2, or 3:0.9:0.1.

In an embodiment, silk fibroin-based protein fragments incorporated intothe tissue fillers described herein have a polydispersity ranging fromabout 1 to about 5.0. In an embodiment, silk fibroin-based proteinfragments incorporated into the tissue fillers described herein have apolydispersity ranging from about 1.5 to about 3.0. In an embodiment,silk fibroin-based protein fragments incorporated into the tissuefillers described herein have a polydispersity ranging from about 1 toabout 1.5. In an embodiment, silk fibroin-based protein fragmentsincorporated into the tissue fillers described herein have apolydispersity ranging from about 1.5 to about 2.0. In an embodiment,silk fibroin-based protein fragments incorporated into the tissuefillers described herein have a polydispersity ranging from about 2.0 toabout 2.5. In an embodiment, a composition of the present disclosurehaving pure silk fibroin-based protein fragments, has a polydispersityranging from about is 2.0 to about 3.0. In an embodiment, a compositionof the present disclosure having pure silk fibroin-based proteinfragments, has a polydispersity ranging from about is 2.5 to about 3.0.

In an embodiment, a tissue filler described herein that includes SPF hasnon-detectable levels of LiBr residuals. In an embodiment, the amount ofthe LiBr residuals in a tissue filler described herein that includes SPFis between 10 ppm and 1000 ppm. In an embodiment, the amount of the LiBrresiduals in a tissue filler described herein that includes SPF isbetween 10 ppm and 300 ppm. In an embodiment, the amount of the LiBrresiduals in a tissue filler described herein that includes SPF is lessthan 25 ppm. In an embodiment, the amount of the LiBr residuals in atissue filler described herein that includes SPF is less than 50 ppm. Inan embodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 75 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 100 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 200 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 300 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 400 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 500 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 600 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 700 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 800 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 900 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is less than 1000 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 500 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 450 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 400 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 350 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 300 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 250 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 200 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 150 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 100 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is 100 ppm to 200 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is 200 ppm to 300 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is 300 ppm to 400 ppm. In anembodiment, the amount of the LiBr residuals in a tissue fillerdescribed herein that includes SPF is 400 ppm to 500 ppm.

In an embodiment, a tissue filler described herein that includes SPFhaving pure silk fibroin-based protein fragments, has non-detectablelevels of Na₂CO₃ residuals. In an embodiment, the amount of the Na₂CO₃residuals in a tissue filler described herein that includes SPF is lessthan 100 ppm. In an embodiment, the amount of the Na₂CO₃ residuals in atissue filler described herein that includes SPF is less than 200 ppm.In an embodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 300 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 400 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 500 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 600 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 700 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 800 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 900 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is less than 1000 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 500 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 450 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 400 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 350 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 300 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 250 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 200 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 150 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is non-detectable to 100 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is 100 ppm to 200 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is 200 ppm to 300 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is 300 ppm to 400 ppm. In anembodiment, the amount of the Na₂CO₃ residuals in a tissue fillerdescribed herein that includes SPF is 400 ppm to 500 ppm.

In an embodiment, the water solubility of pure silk fibroin-basedprotein fragments of the present disclosure is 50 to 100%. In anembodiment, the water solubility of pure silk fibroin-based proteinfragments of the present disclosure is 60 to 100%. In an embodiment, thewater solubility of pure silk fibroin-based protein fragments of thepresent disclosure is 70 to 100%. In an embodiment, the water solubilityof pure silk fibroin-based protein fragments of the present disclosureis 80 to 100%. In an embodiment, the water solubility is 90 to 100%. Inan embodiment, the silk fibroin-based fragments of the presentdisclosure are non-soluble in aqueous solutions.

In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 50 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 60 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 70 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 80 to 100%.In an embodiment, the solubility of pure silk fibroin-based proteinfragments of the present disclosure in organic solutions is 90 to 100%.In an embodiment, the silk fibroin-based fragments of the presentdisclosure are non-soluble in organic solutions.

Methods of making silk protein fragments used in the compositions of thepresent disclosure are demonstrated in U.S. Patent ApplicationPublication Nos. 2015/00933340, 2015/0094269, 2016/0193130,2016/0022560, 2016/0022561, 2016/0022562, 2016/0022563, and2016/0222579, 2016/0281294, and U.S. Pat. Nos. 9,187,538, 9,522,107,9,517,191, 9,522,108, 9,511,012, and 9,545.369, the entirety of whichare incorporated herein by reference. However, an exemplary method isdemonstrated in FIG. 1, which is a flow chart showing variousembodiments for producing pure silk fibroin-based protein fragments(SPFs) of the present disclosure. It should be understood that not allof the steps illustrated are necessarily required to fabricate all silksolutions of the present disclosure. As illustrated in FIG. 1, step A,cocoons (heat-treated or non-heat-treated), silk fibers, silk powder orspider silk can be used as the silk source. If starting from raw silkcocoons from Bombyx mori, the cocoons can be cut into small pieces, forexample pieces of approximately equal size, step B1. The raw silk isthen extracted and rinsed to remove any sericin, step C1a. This resultsin substantially sericin free raw silk. In an embodiment, water isheated to a temperature between 84° C. and 100° C. (ideally boiling) andthen Na₂CO₃ (sodium carbonate) is added to the boiling water until theNa₂CO₃ is completely dissolved. The raw silk is added to the boilingwater/Na₂CO₃ (100° C.) and submerged for approximately 15-90 minutes,where boiling for a longer time results in smaller silk proteinfragments. In an embodiment, the water volume equals about 0.4× raw silkweight and the Na₂CO₃ volume equals about 0.848× raw silk weight. In anembodiment, the water volume equals 0.1× raw silk weight and the Na₂CO₃volume is maintained at 2.12 g/L. This is demonstrated in FIG. 6 andFIG. 7: silk mass (x-axis) was varied in the same volume of extractionsolution (i.e., the same volume of water and concentration of Na₂CO₃)achieving sericin removal (substantially sericin free) as demonstratedby an overall silk mass loss of 26 to 31 percent (y-axis). Subsequently,the water dissolved Na₂CO₃ solution is drained and excess water/Na₂CO₃is removed from the silk fibroin fibers (e.g., ring out the fibroinextract by hand, spin cycle using a machine, etc.). The resulting silkfibroin extract is rinsed with warm to hot water to remove any remainingadsorbed sericin or contaminate, typically at a temperature range ofabout 40° C. to about 80° C., changing the volume of water at least once(repeated for as many times as required). The resulting silk fibroinextract is a substantially sericin-depleted silk fibroin. In anembodiment, the resulting silk fibroin extract is rinsed with water at atemperature of about 60° C. In an embodiment, the volume of rinse waterfor each cycle equals 0.1 L to 0.2 L× raw silk weight. It may beadvantageous to agitate, turn or circulate the rinse water to maximizethe rinse effect. After rinsing, excess water is removed from theextracted silk fibroin fibers (e.g., ring out fibroin extract by hand orusing a machine). Alternatively, methods known to one skilled in the artsuch as pressure, temperature, or other reagents or combinations thereofmay be used for the purpose of sericin extraction. Alternatively, thesilk gland (100% sericin free silk protein) can be removed directly froma worm. This would result in liquid silk protein, without any alterationof the protein structure, free of sericin.

The extracted fibroin fibers are then allowed to dry completely. Oncedry, the extracted silk fibroin is dissolved using a solvent added tothe silk fibroin at a temperature between ambient and boiling, step C1b.In an embodiment, the solvent is a solution of Lithium bromide (LiBr)(boiling for LiBr is 140° C.). Alternatively, the extracted fibroinfibers are not dried but wet and placed in the solvent; solventconcentration can then be varied to achieve similar concentrations as towhen adding dried silk to the solvent. The final concentration of LiBrsolvent can range from 0.1 M to 9.3 M. FIG. 8 is a table summarizing theMolecular Weights of silk dissolved from different concentrations ofLithium Bromide (LiBr) and from different extraction and dissolutionsizes. Complete dissolution of the extracted fibroin fibers can beachieved by varying the treatment time and temperature along with theconcentration of dissolving solvent. Other solvents may be usedincluding, but not limited to, phosphate phosphoric acid, calciumnitrate, calcium chloride solution or other concentrated aqueoussolutions of inorganic salts. To ensure complete dissolution, the silkfibers should be fully immersed within the already heated solventsolution and then maintained at a temperature ranging from about 60° C.to about 140° C. for 1-168 hrs. In an embodiment, the silk fibers shouldbe fully immersed within the solvent solution and then placed into a dryoven at a temperature of about 100° C. for about 1 hour.

The temperature at which the silk fibroin extract is added to the LiBrsolution (or vice versa) has an effect on the time required tocompletely dissolve the fibroin and on the resulting molecular weightand polydispersity of the final SPF mixture solution. In an embodiment,silk solvent solution concentration is less than or equal to 20% w/v. Inaddition, agitation during introduction or dissolution may be used tofacilitate dissolution at varying temperatures and concentrations. Thetemperature of the LiBr solution will provide control over the silkprotein fragment mixture molecular weight and polydispersity created. Inan embodiment, a higher temperature will more quickly dissolve the silkoffering enhanced process scalability and mass production of silksolution. In an embodiment, using a LiBr solution heated to atemperature between 80° C.-140° C. reduces the time required in an ovenin order to achieve full dissolution. Varying time and temperature at orabove 60° C. of the dissolution solvent will alter and control the MWand polydispersity of the SPF mixture solutions formed from the originalmolecular weight of the native silk fibroin protein.

Alternatively, whole cocoons may be placed directly into a solvent, suchas LiBr, bypassing extraction, step B2. This requires subsequentfiltration of silk worm particles from the silk and solvent solution andsericin removal using methods know in the art for separating hydrophobicand hydrophilic proteins such as a column separation and/orchromatography, ion exchange, chemical precipitation with salt and/orpH, and or enzymatic digestion and filtration or extraction, all methodsare common examples and without limitation for standard proteinseparation methods, step C2. Non-heat treated cocoons with the silkwormremoved, may alternatively be placed into a solvent such as LiBr,bypassing extraction. The methods described above may be used forsericin separation, with the advantage that non-heat treated cocoonswill contain significantly less worm debris.

Dialysis may be used to remove the dissolution solvent from theresulting dissolved fibroin protein fragment solution by dialyzing thesolution against a volume of water, step E1. Pre-filtration prior todialysis is helpful to remove any debris (i.e., silk worm remnants) fromthe silk and LiBr solution, step D. In one example, a 3 μm or 5 μmfilter is used with a flow-rate of 200-300 mL/min to filter a 0.1% to1.0% silk-LiBr solution prior to dialysis and potential concentration ifdesired. A method disclosed herein, as described above, is to use timeand/or temperature to decrease the concentration from 9.3 M LiBr to arange from 0.1 M to 9.3 M to facilitate filtration and downstreamdialysis, particularly when considering creating a scalable processmethod. Alternatively, without the use of additional time or temperate,a 9.3 M LiBr-silk protein fragment solution may be diluted with water tofacilitate debris filtration and dialysis. The result of dissolution atthe desired time and temperate filtration is a translucent particle-freeroom temperature shelf-stable silk protein fragment-LiBr solution of aknown MW and polydispersity. It is advantageous to change the dialysiswater regularly until the solvent has been removed (e.g., change waterafter 1 hour, 4 hours, and then every 12 hours for a total of 6 waterchanges). The total number of water volume changes may be varied basedon the resulting concentration of solvent used for silk proteindissolution and fragmentation. After dialysis, the final silk solutionmaybe further filtered to remove any remaining debris (i.e., silk wormremnants).

Alternatively, Tangential Flow Filtration (TFF), which is a rapid andefficient method for the separation and purification of biomolecules,may be used to remove the solvent from the resulting dissolved fibroinsolution, step E2. TFF offers a highly pure aqueous silk proteinfragment solution and enables scalability of the process in order toproduce large volumes of the solution in a controlled and repeatablemanner. The silk and LiBr solution may be diluted prior to TFF (20% downto 0.1% silk in either water or LiBr). Pre-filtration as described aboveprior to TFF processing may maintain filter efficiency and potentiallyavoids the creation of silk gel boundary layers on the filter's surfaceas the result of the presence of debris particles. Pre-filtration priorto TFF is also helpful to remove any remaining debris (i.e., silk wormremnants) from the silk and LiBr solution that may cause spontaneous orlong-term gelation of the resulting water only solution, step D. TFF,recirculating or single pass, may be used for the creation of water-silkprotein fragment solutions ranging from 0.1% silk to 30.0% silk (morepreferably, 0.1%-6.0% silk). Different cutoff size TFF membranes may berequired based upon the desired concentration, molecular weight andpolydispersity of the silk protein fragment mixture in solution.Membranes ranging from 1-100 kDa may be necessary for varying molecularweight silk solutions created for example by varying the length ofextraction boil time or the time and temperate in dissolution solvent(e.g., LiBr). In an embodiment, a TFF 5 or 10 kDa membrane is used topurify the silk protein fragment mixture solution and to create thefinal desired silk-to-water ratio. As well, TFF single pass, TFF, andother methods known in the art, such as a falling film evaporator, maybe used to concentrate the solution following removal of the dissolutionsolvent (e.g., LiBr) (with resulting desired concentration ranging from0.1% to 30% silk). This can be used as an alternative to standard HFIPconcentration methods known in the art to create a water-based solution.A larger pore membrane could also be utilized to filter out small silkprotein fragments and to create a solution of higher molecular weightsilk with and/or without tighter polydispersity values. FIG. 5 is atable summarizing Molecular Weights for some embodiments of silk proteinsolutions of the present disclosure. Silk protein solution processingconditions were as follows: 100° C. extraction for 20 min, roomtemperature rinse, LiBr in 60° C. oven for 4-6 hours. TFF processingconditions for water-soluble films were as follows: 100° C. extractionfor 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for 60 min. FIGS.12-23 further demonstrate manipulation of extraction time, LiBrdissolution conditions, and TFF processing and resultant examplemolecular weights and polydispersities. These examples are not intendedto be limiting, but rather to demonstrate the potential of specifyingparameters for specific molecular weight silk fragment solutions.

An assay for LiBr and Na₂CO₃ detection was performed using an HPLCsystem equipped with evaporative light scattering detector (ELSD). Thecalculation was performed by linear regression of the resulting peakareas for the analyte plotted against concentration. More than onesample of a number of formulations of the present disclosure was usedfor sample preparation and analysis. Generally, four samples ofdifferent formulations were weighed directly in a 10 mL volumetricflask. The samples were suspended in 5 mL of 20 mM ammonium formate (pH3.0) and kept at 2-8° C. for 2 hours with occasional shaking to extractanalytes from the film. After 2 hours the solution was diluted with 20mM ammonium formate (pH 3.0). The sample solution from the volumetricflask was transferred into HPLC vials and injected into the HPLC-ELSDsystem for the estimation of sodium carbonate and lithium bromide.

The analytical method developed for the quantitation of Na₂CO₃ and LiBrin silk protein formulations was found to be linear in the range 10-165sg/mL, with RSD for injection precision as 2% and 1% for area and 0.38%and 0.19% for retention time for sodium carbonate and lithium bromiderespectively. The analytical method can be applied for the quantitativedetermination of sodium carbonate and lithium bromide in silk proteinformulations.

The final silk protein fragment solution is pure silk protein fragmentsand water with PPM to undetectable levels of particulate debris and/orprocess contaminants, including LiBr and Na₂CO₃. FIG. 3 and FIG. 4 aretables summarizing LiBr and Na₂CO₃ concentrations in solutions of thepresent disclosure. In FIG. 3, the processing conditions included 100°C. extraction for 60 min, 60° C. rinse, 100° C. LiBr in 100° C. oven for60 min. TFF conditions including pressure differential and number ofdia-filtration volumes were varied. In FIG. 4, the processing conditionsincluded 100° C. boil for 60 min, 60° C. rinse, LiBr in 60° C. oven for4-6 hours.

Either the silk fragment-water solutions, the lyophilized silk proteinfragment mixture, or any other compositions including SPFs, can besterilized following standard methods in the art not limited tofiltration, heat, radiation or e-beam. It is anticipated that the silkprotein fragment mixture, because of its shorter protein polymer length,will withstand sterilization better than intact silk protein solutionsdescribed in the art. Additionally, silk articles created from the SPFmixtures described herein may be sterilized as appropriate toapplication. For example, an SPF dermal filler loaded with a molecule tobe used in medical applications with an open wound/incision, may besterilized standard methods such as by radiation or e-beam.

FIG. 2 is a flow chart showing various parameters that can be modifiedduring the process of producing a silk protein fragment solution of thepresent disclosure during the extraction and the dissolution steps.Select method parameters may be altered to achieve distinct finalsolution characteristics depending upon the intended use, e.g.,molecular weight and polydispersity. It should be understood that notall of the steps illustrated are necessarily required to fabricate allsilk solutions of the present disclosure.

In an embodiment, a process for producing a silk protein fragmentsolution of the present disclosure includes forming pieces of silkcocoons from the Bombyx mori silk worm; extracting the pieces at about100° C. in a solution of water and Na₂CO₃ for about 60 minutes, whereina volume of the water equals about 0.4× raw silk weight and the amountof Na₂CO₃ is about 0.848× the weight of the pieces to form a silkfibroin extract: triple rinsing the silk fibroin extract at about 60° C.for about 20 minutes per rinse in a volume of rinse water, wherein therinse water for each cycle equals about 0.2 L× the weight of the pieces;removing excess water from the silk fibroin extract; drying the silkfibroin extract; dissolving the dry silk fibroin extract in a LiBrsolution, wherein the LiBr solution is first heated to about 100° C. tocreate a silk and LiBr solution and maintained; placing the silk andLiBr solution in a dry oven at about 100° C. for about 60 minutes toachieve complete dissolution and further fragmentation of the nativesilk protein structure into mixture with desired molecular weight andpolydispersity; filtering the solution to remove any remaining debrisfrom the silkworm; diluting the solution with water to result in a 1%silk solution; and removing solvent from the solution using TangentialFlow Filtration (TFF). In an embodiment, a 10 kDa membrane is utilizedto purify the silk solution and create the final desired silk-to-waterratio. TFF can then be used to further concentrate the pure silksolution to a concentration of 2% silk to water.

Each process step from raw cocoons to dialysis is scalable to increaseefficiency in manufacturing. Whole cocoons are currently purchased asthe raw material, but pre-cleaned cocoons or non-heat treated cocoons,where worm removal leaves minimal debris, have also been used. Cuttingand cleaning the cocoons is a manual process, however for scalabilitythis process could be made less labor intensive by, for example, usingan automated machine in combination with compressed air to remove theworm and any particulates, or using a cutting mill to cut the cocoonsinto smaller pieces. The extraction step, currently performed in smallbatches, could be completed in a larger vessel, for example anindustrial washing machine where temperatures at or in between 60° C. to100° C. can be maintained. The rinsing step could also be completed inthe industrial washing machine, eliminating the manual rinse cycles.Dissolution of the silk in LiBr solution could occur in a vessel otherthan a convection oven, for example a stirred tank reactor. Dialyzingthe silk through a series of water changes is a manual and timeintensive process, which could be accelerated by changing certainparameters, for example diluting the silk solution prior to dialysis.The dialysis process could be scaled for manufacturing by usingsemi-automated equipment, for example a tangential flow filtrationsystem.

Varying extraction (i.e., time and temperature), LiBr (i.e., temperatureof LiBr solution when added to silk fibroin extract or vice versa) anddissolution (i.e., time and temperature) parameters results in solventand silk solutions with different viscosities, homogeneities, andcolors. Increasing the temperature for extraction, lengthening theextraction time, using a higher temperature LiBr solution at emersionand over time when dissolving the silk and increasing the time attemperature (e.g., in an oven as shown here, or an alternative heatsource) all resulted in less viscous and more homogeneous solvent andsilk solutions. While almost all parameters resulted in a viable silksolution, methods that allow complete dissolution to be achieved infewer than 4 to 6 hours are preferred for process scalability.

Molecular weight of the silk protein fragments may be controlled basedupon the specific parameters utilized during the extraction step,including extraction time and temperature; specific parameters utilizedduring the dissolution step, including the LiBr temperature at the timeof submersion of the silk in to the lithium bromide and time that thesolution is maintained at specific temperatures; and specific parametersutilized during the filtration step. By controlling process parametersusing the disclosed methods, it is possible to create SPF mixturesolutions with polydispersity equal to or lower than 2.5 at a variety ofdifferent molecular weight ranging from 1 kDa to 250 kDa, 5 kDa to 200kDa, 5 kDa to 150 kDa, 10 kDa to 150 kDa, or 10 kDa to 80 kDa. Byaltering process parameters to achieve silk solutions with differentmolecular weights, a range of fragment mixture end products, withdesired polydispersity of equal to or less than 2.5 may be targetedbased upon the desired performance requirements. For example, a lowermolecular weight silk film containing a drug may have a faster releaserate compared to a higher molecular weight SPF preparation.Additionally, SPF mixture solutions with a polydispersity of greaterthan 2.5 can be achieved. Further, two solutions with different averagemolecular weights and polydispersities can be mixed to createcombination solutions. Alternatively, a liquid silk gland (100% sericinfree silk protein) that has been removed directly from a worm could beused in combination with any of the SPF mixture solutions of the presentdisclosure. Molecular weight of the pure silk fibroin-based proteinfragment composition was determined using High Pressure LiquidChromatography (HPLC) with a Refractive Index Detector (RID).Polydispersity was calculated using Cirrus GPC Online GPC/SEC SoftwareVersion 3.3 (Agilent).

Parameters were varied during the processing of raw silk cocoons intosilk solution. Varying these parameters affected the MW of the resultingsilk solution. Parameters manipulated included (i) time and temperatureof extraction, (ii) temperature of LiBr, (iii) temperature ofdissolution oven, and (iv) dissolution time. Molecular weight wasdetermined with mass spec as shown in FIGS. 9-25.

Experiments were carried out to determine the effect of varying theextraction time. FIGS. 9-15 are graphs showing these results, and Tables2-8 summarize the results. Below is a summary:

-   -   A sericin extraction time of 30 minutes resulted in larger MW        than a sericin extraction time of 60 minutes    -   MW decreases with time in the oven    -   140° C. LiBr and oven resulted in the low end of the confidence        interval to be below a MW of 9500 Da    -   30 min extraction at the 1 hour and 4 hour time points have        undigested silk    -   30 min extraction at the 1 hour time point resulted in a        significantly high molecular weight with the low end of the        confidence interval being 35,000 Da    -   The range of MW reached for the high end of the confidence        interval was 18000 to 216000 Da (important for offering        solutions with specified upper limit)

TABLE 2 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. Lithium Bromide (LiBr) and 100° C. Oven Dissolution(Oven/ Dissolution Time was varied). Boil Oven Standard Confidence TimeTime Average Mw deviation Interval PD 30 1 57247 12780 35093 93687 1.6360 1 31520 1387 11633 85407 2.71 30 4 40973 2632 14268 117658 2.87 60 425082 1248 10570 59803 2.38 30 6 25604 1405 10252 63943 2.50 60 6 209801262 10073 43695 2.08

TABLE 3 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, boiling Lithium Bromide (LiBr) and 60° C. Oven Dissolutionfor 4 hr. Average Standard Confidence Sample Boil Time Mw deviationInterval PD 30 min, 4 hr 30 49656 4580 17306 142478 2.87 60 min, 4 hr 6030042 1536 11183 80705 2.69

TABLE 4 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 60° C. Lithium Bromide (LiBi) and 60° C. Oven Dissolution(Oven/ Dissolution Time was varied). Boil Oven Average StandardConfidence Sample Time Time Mw deviation Interval PD 30 min, 1 hr 30 158436 22201 153809 2.63 60 min, 1 hr 60 1 31700 11931 84224 2.66 30 min,4 hr 30 4 61956.5 13337 21463 178847 2.89 60 min, 4 hr 60 4 25578.5 24469979 65564 2.56

TABLE 5 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 80° C. Lithium Bromide (LiBr) and 80° C. Oven Dissolutionfor 6 hr. Average Standard Confidence Sample Boil Time Mw deviationInterval PD 30 min, 6 hr 30 63510 18693 215775 3.40 60 min, 6 hr 6025164 238 9637 65706 2.61

TABLE 6 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 80° C. Lithium Bromide (LiBr) and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average StandardConfidence Sample Time Time Mw deviation Interval PD 30 min, 4 hr 30 459202 14028 19073 183760 3.10 60 min, 4 hr 60 4 26312.5 637 10266 674422.56 30 min, 6 hr 30 6 46824 18076 121293 2.59 60 min, 6 hr 60 6 2635310168 68302 2.59

TABLE 7 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 100° C. Lithium Bromide (LiBr) and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average StandardConfidence Sample Time Time Mw deviation Interval PD 30 min, 4 hr 30 447853 19758 115900 2.42 60 min, 4 hr 60 4 25082 1248 10520 59804 2.38 30min, 6 hr 30 6 55421 8992 19153 160366 2.89 60 min, 6 hr 60 6 20980 126210073 43694 2.08

TABLE 8 The effect of extraction time (30 min vs 60 min) on molecularweight of silk processed under the conditions of 100° C. ExtractionTemperature, 140° C. Lithium Bromide (LiBr) and 140° C. Oven Dissolution(Oven/Dissolution Time was varied). Boil Oven Average StandardConfidence Sample Time Time Mw deviation Interval PD 30 min, 4 hr 30 49024.5 1102 4493 18127 2.00865 60 min, 4 hr 60 4 15548 6954 34762 2.235830 min, 6 hr 30 6 13021 5987 28319 2.1749 60 min, 6 hr 60 6 10888 536422100 2.0298

Experiments were carried out to determine the effect of varying theextraction temperature. FIG. 16 is a graph showing these results, andTable 9 summarizes the results. Below is a summary:

-   -   Sericin extraction at 90° C. resulted in higher MW than sericin        extraction at 100° C. extraction    -   Both 90° C. and 100° C. show decreasing MW over time in the oven

TABLE 9 The effect of extraction temperature (90° C. vs. 100° C.) onmolecular weight of silk processed under the conditions of 60 min.Extraction Temperature, 100° C. Lithium Bromide (LiBr) and 100° C. OvenDissolution (Oven/Dissolution Time was varied). Boil Oven AverageStandard Confidence Sample Time Time Mw deviation Interval PD  90° C., 4hr 60 4 37308 4204 13368 104119 2.79 100° C., 4 hr 60 4 25082 1248 1052059804 2.38  90° C., 6 hr 60 6 34224 1135 12717 92100 2.69 100° C., 6 hr60 6 20980 1262 10073 43694 2.08

Experiments were carried out to determine the effect of varying theLithium Bromide (LiBr) temperature when added to silk. FIGS. 17-18 aregraphs showing these results and Tables 10-11 summarize the results.Below is a summary:

-   -   No impact on MW or confidence interval (all CI˜10500-6500 Da)    -   Studies illustrated that the temperature of LiBr-silk        dissolution, as LiBr is added and begins dissolving, rapidly        drops below the original LiBr temperature due to the majority of        the mass being silk at room temp

TABLE 10 The effect of Lithium Bromide (LiBr) temperature on molecularweight of silk processed under the conditions of 60 min. ExtractionTime., 100° C. Extraction Temperature and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). LiBr Temp Oven Average StandardConfidence Sample (° C.) Time Mw deviation Interval PD  60° C. LiBr, 601 31700 11931 84223 2.66 1 hr 100° C. LiBr, 100 1 27907 200 10735 725522.60 1 hr RT LiBr, RT 4 29717 1082 10789 79119 2.71 4 hr  60° C. LiBr,60 4 25578 2445 9978 65564 2.56 4 hr  80° C. LiBr, 80 4 26312 637 1026567441 2.56 4 hr 100° C. LiBr, 100 4 27681 1729 11279 67931 2.45 4 hrBoil LiBr, Boil 4 30042 1535 11183 80704 2.69 4 hr RT LiBr, RT 6 265431893 10783 65332 2.46 6 hr  80° C. LiBr, 80 6 26353 10167 68301 2.59 6hr 100° C. LiBr, 100 6 27150 916 11020 66889 2.46 6 hr

TABLE 11 The effect of Lithium Bromide (LiBr) temperature on molecularweight of silk processed under the conditions of 30 min. ExtractionTime, 100° C. Extraction Temperature and 60° C. Oven Dissolution(Oven/Dissolution Time was varied). LiBr Temp Oven Average StandardConfidence Sample (° C.) Time Mw deviation Interval PD  60° C. LiBr, 604 61956 13336 21463 178847 2.89 4 hr  80° C. LiBr, 80 4 59202 1402719073 183760 3.10 4 hr 100° C. LiBr, 100 4 47853 19757 115899 2.47 4 hr 80° C. LiBr, 80 6 46824 18075 121292 2.59 6 hr 100° C. LiBr, 100 655421 8991 19152 160366 2.89 6 hr

Experiments were carried out to determine the effect of oven/dissolutiontemperature. FIGS. 19-23 are graphs showing these results, and Tables12-16 summarize the results. Below is a summary:

-   -   Oven temperature has less of an effect on 60 min extracted silk        than 30 min extracted silk. Without wishing to be bound by        theory, it is believed that the 30 min silk is less degraded        during extraction and therefore the oven temperature has more of        an effect on the larger MW, less degraded portion of the silk.    -   For 60° C. vs. 140° C. oven the 30 min extracted silk showed a        very significant effect of lower MW at higher oven temp, while        60 min extracted silk had an effect but much less    -   The 140° C. oven resulted in a low end in the confidence        interval at ˜6000 Da

TABLE 12 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 100° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied) Boil Oven Temp Oven Average StandardConfidence Time (° C.) Time Mw deviation Interval PD 30 60 4 47853 19758115900 2.42 30 100 4 40973 2632 14268 117658 2.87 30 60 6 55421 899219153 160366 2.89 30 100 6 25604 1405 10252 63943 2.50

TABLE 13 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 100° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven Average StandardConfidence Time (° C.) Time Mw deviation Interval PD 60 60 1 27908 20010735 72552 2.60 60 100 1 31520 1387 11633 85407 2.71 60 60 4 27681 173011279 72552 2.62 60 100 4 25082 1248 10520 59803 2.38 60 60 6 27150 91611020 66889 2.46 60 100 6 20980 1262 10073 43695 2.08

TABLE 14 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 140° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven Average StandardConfidence Time (° C.) Time Mw deviation Interval PD 60 60 4 30042 153611183 80705 2.69 60 140 4 15548 7255 33322 2.14

TABLE 15 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 30 min. Extraction Time, and 140° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven Average StandardConfidence Time (° C.) Time Mw deviation Interval PD 30 60 4 49656 458017306 142478 2.87 30 140 4 9025 1102 4493 18127 2.01 30 60 6 59383 1164017641 199889 3.37 30 140 6 13021 5987 28319 2.17

TABLE 16 The effect of oven/dissolution temperature on molecular weightof silk processed under the conditions of 100° C. ExtractionTemperature, 60 min. Extraction Time, and 80° C. Lithium Bromide (LiBr)(Oven/Dissolution Time was varied). Boil Oven Temp Oven Average StandardConfidence Time (° C.) Time Mw deviation Interval PD 60 60 4 26313 63710266 67442 2.56 60 80 4 30308 4293 12279 74806 2.47 60 60 6 26353 1016868302 2.59 60 80 6 25164 238 9637 65706 2.61

In an embodiment, the methods disclosed herein result in a solution withcharacteristics that can be controlled during manufacturing, including,but not limited to: MW—may be varied by changing extraction and/ordissolution time and temp (e.g., LiBr temperature), pressure, andfiltration (e.g., size exclusion chromatography); Structure—removal orcleavage of heavy or light chain of the fibroin protein polymer;Purity—hot water rinse temperature for improved sericin removal orfilter capability for improved particulate removal that adverselyaffects shelf stability of the silk fragment protein mixture solution;Color—the color of the solution can be controlled with, for example,LiBr temp and time; Viscosity; Clarity; and Stability of solution. Theresultant pH of the solution is typically about 7 and can be alteredusing an acid or base as appropriate to storage requirements.

The above-described SPF mixture solutions may be utilized to produce SPFcontaining tissue fillers, as described herein.

A method for preparing an aqueous solution of pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from about 1 kDa to about 250 kDa includes the steps of:degumming a silk source by adding the silk source to a boiling (100° C.)aqueous solution of sodium carbonate for a treatment time of betweenabout 30 minutes to about 60 minutes; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 60° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in an ovenhaving a temperature of about 140° C. for a period of at least 1 hour;removing the lithium bromide from the silk fibroin extract; andproducing an aqueous solution of silk protein fragments, the aqueoussolution comprising: fragments having an average weight averagemolecular weight ranging from about 1 kDa to about 250 kDa, and whereinthe aqueous solution of pure silk fibroin-based protein fragmentscomprises a polydispersity of between about 1.5 and about 3.0. Themethod may further comprise drying the silk fibroin extract prior to thedissolving step. The aqueous solution of pure silk fibroin-based proteinfragments may comprise lithium bromide residuals of less than 300 ppm asmeasured using a high-performance liquid chromatography lithium bromideassay. The aqueous solution of pure silk fibroin-based protein fragmentsmay comprise sodium carbonate residuals of less than 100 ppm as measuredusing a high-performance liquid chromatography sodium carbonate assay.The method may further comprise adding a therapeutic agent to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding a molecule selected from one of anantioxidant or an enzyme to the aqueous solution of pure silkfibroin-based protein fragments. The method may further comprise addinga vitamin to the aqueous solution of pure silk fibroin-based proteinfragments. The vitamin may be vitamin C or a derivative thereof. Theaqueous solution of pure silk fibroin-based protein fragments may belyophilized. The method may further comprise adding an alpha hydroxyacid to the aqueous solution of pure silk fibroin-based proteinfragments. The alpha hydroxy acid may be selected from the groupconsisting of glycolic acid, lactic acid, tartaric acid and citric acid.The method may further comprise adding hyaluronic acid or its salt format a concentration of about 0.5% to about 10.0% to the aqueous solutionof pure silk fibroin-based protein fragments. The method may furthercomprise adding at least one of zinc oxide or titanium dioxide. A filmmay be fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The film may comprise fromabout 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivativethereof. The film may have a water content ranging from about 2.0 wt. %to about 20.0 wt. %. The film may comprise from about 30.0 wt. % toabout 99.5 wt. % of pure silk fibroin-based protein fragments. A gel maybe fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The gel may comprise fromabout 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivativethereof. The gel may have a silk content of at least 2% and a vitamincontent of at least 20%.

A method for preparing an aqueous solution of pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from about 5 kDa to about 150 kDa includes the steps of:degumming a silk source by adding the silk source to a boiling (100° C.)aqueous solution of sodium carbonate for a treatment time of betweenabout 30 minutes to about 60 minutes; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 60° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in an ovenhaving a temperature of about 140° C. for a period of at least 1 hour;removing the lithium bromide from the silk fibroin extract; andproducing an aqueous solution of silk protein fragments, the aqueoussolution comprising: fragments having an average weight averagemolecular weight ranging from about 5 kDa to about 150 kDa, and whereinthe aqueous solution of pure silk fibroin-based protein fragmentscomprises a polydispersity of between about 1.5 and about 3.0. Themethod may further comprise drying the silk fibroin extract prior to thedissolving step. The aqueous solution of pure silk fibroin-based proteinfragments may comprise lithium bromide residuals of less than 300 ppm asmeasured using a high-performance liquid chromatography lithium bromideassay. The aqueous solution of pure silk fibroin-based protein fragmentsmay comprise sodium carbonate residuals of less than 100 ppm as measuredusing a high-performance liquid chromatography sodium carbonate assay.The method may further comprise adding a therapeutic agent to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding a molecule selected from one of anantioxidant or an enzyme to the aqueous solution of pure silkfibroin-based protein fragments. The method may further comprise addinga vitamin to the aqueous solution of pure silk fibroin-based proteinfragments. The vitamin may be vitamin C or a derivative thereof. Theaqueous solution of pure silk fibroin-based protein fragments may belyophilized. The method may further comprise adding an alpha hydroxyacid to the aqueous solution of pure silk fibroin-based proteinfragments. The alpha hydroxy acid may be selected from the groupconsisting of glycolic acid, lactic acid, tartaric acid and citric acid.The method may further comprise adding hyaluronic acid or its salt format a concentration of about 0.5% to about 10.0% to the aqueous solutionof pure silk fibroin-based protein fragments. The method may furthercomprise adding at least one of zinc oxide or titanium dioxide. A filmmay be fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The film may comprise fromabout 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivativethereof. The film may have a water content ranging from about 2.0 wt. %to about 20.0 wt. %. The film may comprise from about 30.0 wt. % toabout 99.5 wt. % of pure silk fibroin-based protein fragments. A gel maybe fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The gel may comprise fromabout 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivativethereof. The gel may have a silk content of at least 2% and a vitamincontent of at least 20%.

A method for preparing an aqueous solution of pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from about 6 kDa to about 17 kDa includes the steps of:degumming a silk source by adding the silk source to a boiling (100° C.)aqueous solution of sodium carbonate for a treatment time of betweenabout 30 minutes to about 60 minutes; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 60° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in an ovenhaving a temperature of about 140° C. for a period of at least 1 hour;removing the lithium bromide from the silk fibroin extract; andproducing an aqueous solution of silk protein fragments, the aqueoussolution comprising: fragments having an average weight averagemolecular weight ranging from about 6 kDa to about 17 kDa. and whereinthe aqueous solution of pure silk fibroin-based protein fragmentscomprises a polydispersity of between about 1.5 and about 3.0. Themethod may further comprise drying the silk fibroin extract prior to thedissolving step. The aqueous solution of pure silk fibroin-based proteinfragments may comprise lithium bromide residuals of less than 300 ppm asmeasured using a high-performance liquid chromatography lithium bromideassay. The aqueous solution of pure silk fibroin-based protein fragmentsmay comprise sodium carbonate residuals of less than 100 ppm as measuredusing a high-performance liquid chromatography sodium carbonate assay.The method may further comprise adding a therapeutic agent to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding a molecule selected from one of anantioxidant or an enzyme to the aqueous solution of pure silkfibroin-based protein fragments. The method may further comprise addinga vitamin to the aqueous solution of pure silk fibroin-based proteinfragments. The vitamin may be vitamin C or a derivative thereof. Theaqueous solution of pure silk fibroin-based protein fragments may belyophilized. The method may further comprise adding an alpha hydroxyacid to the aqueous solution of pure silk fibroin-based proteinfragments. The alpha hydroxy acid may be selected from the groupconsisting of glycolic acid, lactic acid, tartaric acid and citric acid.The method may further comprise adding hyaluronic acid or its salt format a concentration of about 0.5% to about 10.0% to the aqueous solutionof pure silk fibroin-based protein fragments. The method may furthercomprise adding at least one of zinc oxide or titanium dioxide. A filmmay be fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The film may comprise fromabout 1.0 wt. % to about 50.0 wt. % of vitamin C or a derivativethereof. The film may have a water content ranging from about 2.0 wt. %to about 20.0 wt. %. The film may comprise from about 30.0 wt. % toabout 99.5 wt. % of pure silk fibroin-based protein fragments. A gel maybe fabricated from the aqueous solution of pure silk fibroin-basedprotein fragments produced by this method. The gel may comprise fromabout 0.5 wt. % to about 20.0 wt. % of vitamin C or a derivativethereof. The gel may have a silk content of at least 2% and a vitamincontent of at least 20%.

A method for preparing an aqueous solution of pure silk fibroin-basedprotein fragments having an average weight average molecular weightranging from about 17 kDa to about 39 kDa includes the steps of: addinga silk source to a boiling (100° C.) aqueous solution of sodiumcarbonate for a treatment time of between about 30 minutes to about 60minutes so as to result in degumming; removing sericin from the solutionto produce a silk fibroin extract comprising non-detectable levels ofsericin; draining the solution from the silk fibroin extract; dissolvingthe silk fibroin extract in a solution of lithium bromide having astarting temperature upon placement of the silk fibroin extract in thelithium bromide solution that ranges from about 80° C. to about 140° C.;maintaining the solution of silk fibroin-lithium bromide in a dry ovenhaving a temperature in the range between about 60° C. to about 100° C.for a period of at least 1 hour; removing the lithium bromide from thesilk fibroin extract; and producing an aqueous solution of pure silkfibroin-based protein fragments, wherein the aqueous solution of puresilk fibroin-based protein fragments comprises lithium bromide residualsof between about 10 ppm and about 300 ppm, wherein the aqueous solutionof silk protein fragments comprises sodium carbonate residuals ofbetween about 10 ppm and about 100 ppm, wherein the aqueous solution ofpure silk fibroin-based protein fragments comprises fragments having anaverage weight average molecular weight ranging from about 17 kDa toabout 39 kDa, and wherein the aqueous solution of pure silkfibroin-based protein fragments comprises a polydispersity of betweenabout 1.5 and about 3.0. The method may further comprise drying the silkfibroin extract prior to the dissolving step. The aqueous solution ofpure silk fibroin-based protein fragments may comprise lithium bromideresiduals of less than 300 ppm as measured using a high-performanceliquid chromatography lithium bromide assay. The aqueous solution ofpure silk fibroin-based protein fragments may comprise sodium carbonateresiduals of less than 100 ppm as measured using a high-performanceliquid chromatography sodium carbonate assay. The method may furthercomprise adding a therapeutic agent to the aqueous solution of pure silkfibroin-based protein fragments. The method may further comprise addinga molecule selected from one of an antioxidant or an enzyme to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding a vitamin to the aqueous solution ofpure silk fibroin-based protein fragments. The vitamin may be vitamin Cor a derivative thereof. The aqueous solution of pure silk fibroin-basedprotein fragments may be lyophilized. The method may further compriseadding an alpha hydroxy acid to the aqueous solution of pure silkfibroin-based protein fragments. The alpha hydroxy acid may be selectedfrom the group consisting of glycolic acid, lactic acid, tartaric acidand citric acid. The method may further comprise adding hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0% to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding at least one of zinc oxide ortitanium dioxide.

A gel may be fabricated from the aqueous solution of pure silkfibroin-based protein fragments produced by this method. The gel maycomprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or aderivative thereof. The gel may have a silk content of at least 2% and avitamin content of at least 20%.

According to aspects illustrated herein, there is disclosed a method forpreparing an aqueous solution of pure silk fibroin-based proteinfragments having an average weight average molecular weight ranging fromabout 39 kDa to about 80 kDa, the method including the steps of: addinga silk source to a boiling (100° C.) aqueous solution of sodiumcarbonate for a treatment time of about 30 minutes so as to result indegumming; removing sericin from the solution to produce a silk fibroinextract comprising non-detectable levels of sericin; draining thesolution from the silk fibroin extract; dissolving the silk fibroinextract in a solution of lithium bromide having a starting temperatureupon placement of the silk fibroin extract in the lithium bromidesolution that ranges from about 80° C. to about 140° C.; maintaining thesolution of silk fibroin-lithium bromide in a dry oven having atemperature in the range between about 60° C. to about 100° C. for aperiod of at least 1 hour; removing the lithium bromide from the silkfibroin extract; and producing an aqueous solution of pure silkfibroin-based protein fragments, wherein the aqueous solution of puresilk fibroin-based protein fragments comprises lithium bromide residualsof between about 10 ppm and about 300 ppm, sodium carbonate residuals ofbetween about 10 ppm and about 100 ppm, fragments having an averageweight average molecular weight ranging from about 40 kDa to about 65kDa, and wherein the aqueous solution of pure silk fibroin-based proteinfragments comprises a polydispersity of between about 1.5 and about 3.0.The method may further comprise drying the silk fibroin extract prior tothe dissolving step. The aqueous solution of pure silk fibroin-basedprotein fragments may comprise lithium bromide residuals of less than300 ppm as measured using a high-performance liquid chromatographylithium bromide assay. The aqueous solution of pure silk fibroin-basedprotein fragments may comprise sodium carbonate residuals of less than100 ppm as measured using a high-performance liquid chromatographysodium carbonate assay. The method may further comprise adding atherapeutic agent to the aqueous solution of pure silk fibroin-basedprotein fragments. The method may further comprise adding a moleculeselected from one of an antioxidant or an enzyme to the aqueous solutionof pure silk fibroin-based protein fragments. The method may furthercomprise adding a vitamin to the aqueous solution of pure silkfibroin-based protein fragments. The vitamin may be vitamin C or aderivative thereof. The aqueous solution of pure silk fibroin-basedprotein fragments may be lyophilized. The method may further compriseadding an alpha hydroxy acid to the aqueous solution of pure silkfibroin-based protein fragments. The alpha hydroxy acid may be selectedfrom the group consisting of glycolic acid, lactic acid, tartaric acidand citric acid. The method may further comprise adding hyaluronic acidor its salt form at a concentration of about 0.5% to about 10.0% to theaqueous solution of pure silk fibroin-based protein fragments. Themethod may further comprise adding at least one of zinc oxide ortitanium dioxide.

A gel may be fabricated from the aqueous solution of pure silkfibroin-based protein fragments produced by this method. The gel maycomprise from about 0.5 wt. % to about 20.0 wt. % of vitamin C or aderivative thereof. The gel may have a silk content of at least 2% and avitamin content of at least 20%.

Hyaluronic Acid and Hyaluronic Acid Gels

A biodegradable polymer component of the present invention ishyaluronate, also known as hyaluronic acid (HA). HA consists ofalternating residues of D-glucuronic acid and N-acetyl-D-glucosamine.This water soluble polymer is naturally found in nearly all tissue,especially in the extracellular matrix, the eyes and synovial fluid ofjoints. HA is commercially available in pure form. Small gel particle HAfillers may be used stimulate natural collagen production that ispresumed to be induced by mechanical stretching of the dermis andactivation of dermal fibroblasts.

HA concentration in the resulting dermal fillers of the inventioncontributes to dermal filler stiffness and longevity. In someembodiments, an increased concentration of HA in the resulting dermalfillers described herein may increase the stiffness and/or longevity ofthe resulting dermal filler as compared to a dermal filler having acomparatively lesser concentration of HA.

In some embodiments, HA incorporated in the tissue fillers describedherein has a molecular weight of 100,000 daltons or greater, 150,000daltons or greater, 1 million daltons or greater, or 2 million daltonsor greater. In some embodiments, HA incorporated in the tissue fillersdescribed herein has a molecular weight of 100,000 daltons or less,150,000 daltons or less, 1 million daltons or less, or 2 million daltonsor less. In some embodiments, the HA incorporated in the tissue fillersdescribed herein has a high molecular weight (e.g., an HA molecularweight of about 1 MDa to about 4 MDa). In some embodiments, the HAincorporated in the tissue fillers described herein has a low molecularweight (e.g., an HA molecular weight of less than about 1 MDa).

In some embodiments, the HA source may be a hyaluronate salt such as,for example, sodium hyaluronate. In some embodiments, the HA iscross-linked. Cross-linked HA can be formulated into a variety ofshapes, such as membranes, gels, semi-gels, sponges, or microspheres. Insome embodiments, the cross-linked HA is in fluid gel form, i.e., ittakes the shape of its container. The viscosity of an HA gel or semi-gelcan be altered by the addition of unconjugated HA and/or hyaluronate.Viscosity can also be tuned by varying the degree of SPF-SPF, SPF-HA,and/or HA-HA cross-linking as described herein. In some embodiment,about 4% to about 12% of the HA may be cross-linked as HA-HA or HA-SPF.

In an embodiment, the SPF compositions described herein may be combinedwith HA to form a tissue filler composition. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 99%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 98%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 97%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 96%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 95%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 94%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 93%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 92%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 91%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 90%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 85%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 80%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 75%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 70%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 65%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 600%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 55%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 50%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 45%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 40%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 35%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 30%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 25%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 20%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 19%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 18%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 17%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 16%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 15%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 14%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 13%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 12%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 11%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 10%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 9%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 8%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 7%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 6%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 5%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 4%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 3%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 2%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 1%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 0.9%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 0.8%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 0.7%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 0.6%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 0.5%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 0.4%.In an embodiment, the percent HA in the tissue filler composition byweight is less than 0.3%. In an embodiment, the percent HA in the tissuefiller composition by weight is less than 0.2%. In an embodiment, thepercent HA in the tissue filler composition by weight is less than 0.1%.In an embodiment, the percent HA in the tissue filler composition byweight is greater than 0.1%. In an embodiment, the percent HA in thetissue filler composition by weight is greater than 0.2%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 0.3%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 0.4%. In an embodiment, thepercent HA in the tissue filler composition by weight is greater than0.5%. In an embodiment, the percent HA in the tissue filler compositionby weight is greater than 0.6%. In an embodiment, the percent HA in thetissue filler composition by weight is greater than 0.7%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 0.8%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 0.9%. In an embodiment, thepercent HA in the tissue filler composition by weight is greater than1%. In an embodiment, the percent HA in the tissue filler composition byweight is greater than 2%. In an embodiment, the percent HA in thetissue filler composition by weight is greater than 3%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 4%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 5%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 6%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 7%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 8%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 9%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 10%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 11%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 12%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 13%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 14%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 15%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 16%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 17%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 18%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 19%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 20%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 25%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 30%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 35%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 40%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 45%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 50%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 55%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 60%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 65%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 70%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 75%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 80%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 85%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 90%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 91%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 92%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 93%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 94%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 95%. In anembodiment, the percent HA in the tissue filler composition by weight isgreater than 96%. In an embodiment, the percent HA in the tissue fillercomposition by weight is greater than 97%. In an embodiment, the percentHA in the tissue filler composition by weight is greater than 98%.

In an embodiment, the percent HA in the tissue filler composition byweight is about 0.1%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 0.2%. In an embodiment, thepercent HA in the tissue filler composition by weight is about 0.3%. Inan embodiment, the percent HA in the tissue filler composition by weightis about 0.4%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 0.5%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 0.6%. In an embodiment,the percent HA in the tissue filler composition by weight is about 0.7%.In an embodiment, the percent HA in the tissue filler composition byweight is about 0.8%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 0.9%. In an embodiment, thepercent HA in the tissue filler composition by weight is about 1%. In anembodiment, the percent HA in the tissue filler composition by weight isabout 2%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 3%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 4%. In an embodiment,the percent HA in the tissue filler composition by weight is about 5%.In an embodiment, the percent HA in the tissue filler composition byweight is about 6%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 7%. In an embodiment, the percentHA in the tissue filler composition by weight is about 8%. In anembodiment, the percent HA in the tissue filler composition by weight isabout 9%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 10%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 11%. In an embodiment,the percent HA in the tissue filler composition by weight is about 12%.In an embodiment, the percent HA in the tissue filler composition byweight is about 13%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 14%. In an embodiment, the percentHA in the tissue filler composition by weight is about 15%. In anembodiment, the percent HA in the tissue filler composition by weight isabout 16%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 17%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 18%. In an embodiment,the percent HA in the tissue filler composition by weight is about 19%.In an embodiment, the percent HA in the tissue filler composition byweight is about 20%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 25%. In an embodiment, the percentHA in the tissue filler composition by weight is about 30%. In anembodiment, the percent HA in the tissue filler composition by weight isabout 35%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 40%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 45%. In an embodiment,the percent HA in the tissue filler composition by weight is about 50%.In an embodiment, the percent HA in the tissue filler composition byweight is about 55%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 60%. In an embodiment, the percentHA in the tissue filler composition by weight is about 65%. In anembodiment, the percent HA in the tissue filler composition by weight isabout 70%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 75%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 80%. In an embodiment,the percent HA in the tissue filler composition by weight is about 85%.In an embodiment, the percent HA in the tissue filler composition byweight is about 90%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 91%. In an embodiment, the percentHA in the tissue filler composition by weight is about 92%. In anembodiment, the percent HA in the tissue filler composition by weight isabout 93%. In an embodiment, the percent HA in the tissue fillercomposition by weight is about 94%. In an embodiment, the percent HA inthe tissue filler composition by weight is about 95%. In an embodiment,the percent HA in the tissue filler composition by weight is about 96%.In an embodiment, the percent HA in the tissue filler composition byweight is about 97%. In an embodiment, the percent HA in the tissuefiller composition by weight is about 98%.

In an embodiment, the percent HA in the tissue filler composition byweight is between about 0.1% to about 1%. In an embodiment, the percentHA in the tissue filler composition by weight is between about 0.5% toabout 1.5%. In an embodiment, the percent HA in the tissue fillercomposition by weight is between about 1% to about 5%. In an embodiment,the percent HA in the tissue filler composition by weight is betweenabout 1.5% to about 5.5%. In an embodiment, the percent HA in the tissuefiller composition by weight is between about 2% to about 6%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 2.5% to about 6.5%. In an embodiment, the percent HA inthe tissue filler composition by weight is between about 3% to about 7%.In an embodiment, the percent HA in the tissue filler composition byweight is between about 3.5% to about 7.5%. In an embodiment, thepercent HA in the tissue filler composition by weight is between about4% to about 8%. In an embodiment, the percent HA in the tissue fillercomposition by weight is between about 4.5% to about 8.5%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 5% to about 9%. In an embodiment, the percent HA in thetissue filler composition by weight is between about 5.5% to about 9.5%.In an embodiment, the percent HA in the tissue filler composition byweight is between about 6% to about 10%. In an embodiment, the percentHA in the tissue filler composition by weight is between about 6.5% toabout 10.5%. In an embodiment, the percent HA in the tissue fillercomposition by weight is between about 7% to about 11%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 7.5% to about 11.5%. In an embodiment, the percent HA inthe tissue filler composition by weight is between about 8% to about12%. In an embodiment, the percent HA in the tissue filler compositionby weight is between about 8.5% to about 12.5%. In an embodiment, thepercent HA in the tissue filler composition by weight is between about9% to about 13%. In an embodiment, the percent HA in the tissue fillercomposition by weight is between about 9.5% to about 13.5%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 10% to about 14%. In an embodiment, the percent HA in thetissue filler composition by weight is between about 10.5% to about14.5%. In an embodiment, the percent HA in the tissue filler compositionby weight is between about 11% to about 15%. In an embodiment, thepercent HA in the tissue filler composition by weight is between about11.5% to about 15.5%. In an embodiment, the percent HA in the tissuefiller composition by weight is between about 12% to about 16%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 12.5% to about 16.5%. In an embodiment, the percent HA inthe tissue filler composition by weight is between about 13% to about17%. In an embodiment, the percent HA in the tissue filler compositionby weight is between about 13.5% to about 17.5%. In an embodiment, thepercent HA in the tissue filler composition by weight is between about14% to about 18%. In an embodiment, the percent HA in the tissue fillercomposition by weight is between about 14.5% to about 18.5%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 15% to about 19%. In an embodiment, the percent HA in thetissue filler composition by weight is between about 15.5% to about19.5%. In an embodiment, the percent HA in the tissue filler compositionby weight is between about 16% to about 20%. In an embodiment, thepercent HA in the tissue filler composition by weight is between about20% to about 30%. In an embodiment, the percent HA in the tissue fillercomposition by weight is between about 30% to about 40%. In anembodiment, the percent HA in the tissue filler composition by weight isbetween about 40% to about 50%. In an embodiment, the percent HA in thetissue filler composition by weight is between about 50% to about 60%.In an embodiment, the percent HA in the tissue filler composition byweight is between about 60% to about 70%. In an embodiment, the percentHA in the tissue filler composition by weight is between about 80% toabout 90%

In some embodiments, the percent HA, by weight, in the tissue fillercompositions described herein is about 1% to about 2%, or about 1% toabout 3%, or about 1% to about 4%, or about 1% to about 5%, or about 1%to about 6%, or about 1% to about 7%, or about 1% to about 8%, or about1% to about 9%, or about 1% to about 10%, or about 1% to about 11%, orabout 1% to about 12%, or about 1% to about 13%, or about 1% to about14%, or about 1% to about 15%, or about 1% to about 16%, or about 1% toabout 17%, or about 1% to about 18%, or about 1% to about 19%, or about1% to about 20%, or about 1% to about 21%, or about 1% to about 22%, orabout 1% to about 23%, or about 1% to about 24%, or about 1% to about25%, or about 1% to about 30%, or about 1% to about 400%, or about 1% toabout 50%, or about 1% to about 60% 0, or about 1% to about 70%, orabout 1% to about 80%, or about 1% to about 95%; or about 10% to about20%, or about 10% to about 25%, or about 10% to about 30%, or about 10%to about 35%, or about 10% to about 40%, or about 10% to about 45%, orabout 10% to about 50%, or about 10% to about 55%, or about 10% to about60%, or about 10% to about 65%, or about 10% to about 70%, or about 10%to about 75%, or about 10% to about 80%, or about 10% to about 85%, orabout 10% to about 900%, or about 10% to about 95%.

In some embodiments, the HA described herein may be acquired fromcommercial sources or may be produced by Streptococcus equi bacteria.

Tissue fillers described herein that include HA may be characterized fortheir in vitro biological activities and in vivo biological activities.For example, in vitro assays may be performed on a portion of the tissuefillers described herein for cell toxicity, resistance to enzymaticdegradation, syringeability (e.g., solution viscosity, injection flowrate, syringe/needle diameter), and/or particle morphology analysis.See, e.g., Park, et al., J. Eur. Acad. Dermatol. Venerol. (2014)28:565-568. In vivo assays may be performed to determine initialmorphological patterns, total remaining filler present, histologicalevaluations, and may include the examination of granuloma formation orcutaneous adverse reactions. See, e.g., Park, et al., J. Eur. Acad.Dermatol. Venerol. (2014) 28:565-568; and Ramot, et al., ToxicologyPathology (2015) 43: 267-271.

Gelation

In an embodiment, silk gels may be provided with a gelation aid. In someembodiments, the gelation aid may be an acid, electricity, mixing,and/or sonication.

In an embodiment, when producing a silk gel, an acid can be added to asilk solution described herein to help facilitate gelation. In anembodiment, when producing a silk gel that includes a neutral or a basicmolecule and/or therapeutic agent, an acid can be added to facilitategelation. In an embodiment, when producing a silk gel, increasing the pH(making the gel more basic) increases the shelf stability of the gel. Inan embodiment, when producing a silk gel, increasing the pH (making thegel more basic) allows for a greater quantity of an acidic molecule tobe loaded into the gel.

In an embodiment, when producing a silk gel, electricity can be passedthrough a silk solution described herein to help facilitate gelation.

In an embodiment, when producing a silk gel, mixing of a silk solutiondescribed herein can be used to help facilitate gelation.

In an embodiment, when producing a silk gel, sonication of a silksolution described herein can be used to help facilitate gelation.

In an embodiment, natural additives may be added to the silk gel tofurther stabilize additives. For example, trace elements such asselenium or magnesium or L-methionine can be used. Further, light-blockcontainers can be added to further increase stability.

In some embodiments, gelation enhancers can be used to accelerate SPFgelation. In some embodiments, an SPF solution can be mixed with purealcohol or aqueous alcohol solution at varied volume ratios accompaniedby mixing, either through stirring, shaking or any other form ofagitation. In some embodiments, this alcohol solution enhancer may thenhave a quantity of an amphiphilic peptide added as a further enhancer ofthe final gel outcome. The extent of acceleration may be heightened orlessened as appropriate by adding a larger or smaller enhancer componentto the system.

In some embodiments, gelation rate may be enhanced by increasing theconcentration of SPF in a solution used for making a gel. Variousmethods can be used to that end, including but not limited to: dialysisof intermediate SPF solution against a buffer incorporating ahygroscopic species such as polyethylene glycol, a lyophilization step,and/or an evaporation step. Increased temperature may also be used as anenhancer of the gelation process. In addition, manipulation ofintermediate silk solution pH by methods including but not limited todirect titration and gas exchange can be used to enhance the gelationprocess. Introduction of select ionic species including calcium andpotassium in particular may also be used to accelerate gelation rate.

In some embodiments, gelation can be helped by the use of nucleatingagents, including organic and inorganic species, both soluble andinsoluble in an SPF intermediate. Nucleating agents can include but arenot limited to peptide sequences which bind silk molecules, previouslygelled silk, and poorly soluble β-sheet rich structures. In someembodiments, a further means of accelerating the gelation process isthrough the introduction of mechanical excitation, which can be impartedthrough a shearing device, ultrasound device, or mechanical mixer.

The time necessary for complete silk solution gelation may vary fromseconds to hours or days, depending on the values of the above mentionedparameters as well as the initial state of aggregation and organizationfound in the SPF solution. The volume fraction of added enhancer mayvary from about 0% to about 99% of the total system volume (i.e., eithercomponent may be added to a large excess of the other or in any relativeconcentration within the interval). The concentration of SPF solutionused can range from about 1% (w/v), to about 20% (w/v), and any otherappropriate range. The enhancer can be added to SPF solution or the SPFsolution can be added to enhancer. The formed SPF hydrogel may befurther chemically or physically cross-linked to gain altered mechanicalproperties.

In some embodiments, an enhancer solution is added to an SPF solution,or vice-versa, the SPF solution having a concentration of SPF of about1% (w/v), about 2% (w/v), about 3% (w/v), about 4% (w/v), about 5%(w/v), about 6% (w/v), about 7% (w/v), about 8% (w/v), about 9% (w/v),about 10% (w/v), about 12% (w/v), about 15% (w/v), about 18% (w/v),about 20% (w/v), about 25% (w/v), or about 30% (w/v). In someembodiments, an enhancer solution is added to an SPF solution, orvice-versa, the SPF solution having a concentration of SPF of at least1% (w/v), at least 2% (w/v), at least 3% (w/v), at least 4% (w/v), atleast 5% (w/v), at least 6% (w/v), at least 7% (w/v), at least 8% (w/v),at least 9% (w/v), at least 10% (w/v), at least 12% (w/v), at least 15%(w/v), at least 18% (w/v), at least 20% (w/v), at least 25% (w/v), or atleast 30% (w/v). In some embodiments, an enhancer solution is added toan SPF solution, or vice-versa, the SPF solution having a concentrationof SPF of about 1% (w/v) to about 5% (w/v), about 1% (w/v) to about 10%(w/v), about 1% (w/v) to about 15% (w/v), about 1% (w/v) to about 20%(w/v), about 1% (w/v) to about 25% (w/v), about 1% (w/v) to about 30%(w/v), about 5% (w/v) to about 10% (w/v), about 5% (w/v) to about 15%(w/v), about 5% (w/v) to about 20% (w/v), about 5% (w/v) to about 25%(w/v), about 5% (w/v) to about 30% (w/v), about 10% (w/v) to about 15%(w/v), about 10% (w/v) to about 20% (w/v), about 10% (w/v) to about 25%(w/v), or about 10% (w/v) to about 30% (w/v).

Gels and Hydrogels—Modifying and Cross-Linking

In some embodiments, the invention provides compositions comprising oneor more hydrogels comprising one or more cross-linked matrix polymers.As used herein, the term “cross-linked” refers to the intermolecularbonds joining the individual polymer molecules, macromolecules, and/ormonomer chains, into a more stable structure like a gel. As such, across-linked matrix polymer has at least one intermolecular bond joiningat least one individual polymer molecule to another one, where the firstindividual polymer molecule can be of similar, or different, chemicalnature to the other. Matrix polymers disclosed herein may becross-linked using dialdehydes and disulfides cross-linking agentsincluding, without limitation, multifunctional PEG-based crosslinkingagents, divinyl sulfones, diglycidyl ethers, and bis-epoxides.Non-limiting examples of SPF, and/or HA, cross-linking agents includedivinyl sulfone (DVS), 1,4-butanediol diglycidyl ether (BDDE), UV light,glutaraldehyde, 1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE),1,2,7,8-diepoxyoctane (DEO), biscarbodiimide (BCD), pentaerythritoltetraglycidyl ether (PETGE), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (HMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, or combinations thereof. Insome embodiments, the HA cross-linking agent may include BDDE or DVS. Insome embodiments, the HA and/or SPF cross-linking agent may be BDDE,DVS, UV light, glutaraldehyde, or a carbodiimide, as described herein.

In some embodiments, the tissue fillers described herein may containresidual cross-linking agent. In some embodiments, the tissue fillersdescribed herein may contain only trace amounts of the cross-linkingagent such as, for example, no greater than about 2 ppm, or no greaterthan about 1.9 ppm, or no greater than about 1.8 ppm, or no greater thanabout 1.7 ppm, or no greater than about 1.6 ppm, or no greater thanabout 1.5 ppm, or no greater than about 1.4 ppm, or no greater thanabout 1.3 ppm, or no greater than about 1.2 ppm, or no greater thanabout 1.1 ppm, or no greater than about 1.0 ppm, or no greater thanabout 0.9 ppm, or no greater than about 0.8 ppm, or no greater thanabout 0.7 ppm, or no greater than about 0.6 ppm, or no greater thanabout 0.5 ppm, or no greater than about 0.4 ppm, or no greater thanabout 0.3 ppm, or no greater than about 0.2 ppm, or no greater thanabout 0.1 ppm. In some embodiments, the tissue fillers described hereinmay contain trace amounts BDDE, but at a concentration no greater thanabout 2 ppm, or no greater than about 1.9 ppm, or no greater than about1.8 ppm, or no greater than about 1.7 ppm, or no greater than about 1.6ppm, or no greater than about 1.5 ppm, or no greater than about 1.4 ppm,or no greater than about 1.3 ppm, or no greater than about 1.2 ppm, orno greater than about 1.1 ppm, or no greater than about 1.0 ppm, or nogreater than about 0.9 ppm, or no greater than about 0.8 ppm, or nogreater than about 0.7 ppm, or no greater than about 0.6 ppm, or nogreater than about 0.5 ppm, or no greater than about 0.4 ppm, or nogreater than about 0.3 ppm, or no greater than about 0.2 ppm, or nogreater than about 0.1 ppm. As understood by a person having ordinaryskill in the art, the amount of residual cross-linking agent present ina particular tissue filler sample may be determined by gaschromatography-mass spectrometry.

In some embodiments, the tissue fillers described herein may include amatrix that may include an SPF matrix portion and an HA matrix portion,where the SPF matrix portion includes a mixture of cross-linked andnon-cross-linked SPF and the HA matrix portion includes a mixture ofcross-linked and non-cross-linked HA.

In some embodiments, the tissue fillers of the invention include linkermodified HA. In some embodiments, the tissue fillers of the inventioninclude linker modified SPF. Bifunctional cross-linkers can react atboth ends to connect two different HA molecules, two different SPFmolecules, or an HA molecule with an SPF molecule. In some embodiments,the cross-linker bonds with an HA molecule only at one end, leaving theother end pendant. In some embodiments, the cross-linker bonds with anSPF molecule only at one end, leaving the other end pendant.

As used herein, the degree of modification (MoD) can be defined as (seefor example J. Kablik et al., Dermatol Surg, 2009 (35): 302-312):

Total % Degree of Modification=% Crosslink+% Pendant

In order to determine the MoD, it can also be defined as (see forexample L. Kenne et al., Carbohydrate Polymers, 2013 (91): 410-418):

${MoD} = \frac{n_{{linked}\mspace{14mu}{crosslinkers}}}{n_{{HA}\mspace{14mu}{disaccharides}} + n_{{SPF}\mspace{14mu}{repeating}\mspace{14mu}{units}}}$

where n_(linked crosslinkers) is the number of linked cross-linkermolecules, n_(HA disaccharides) is the number or disaccharides in HA,and n_(SPF repeating units) is the number of repeating units in SPF.These numbers can be determined by NMR using characteristic chemicalshifts of crosslinker, HA, and SPF.

In some embodiments, the MoD is between about 1% and 25%, between about2% and about 20%, or between about 3.5% and about 17.5%. In someembodiments, the MoD is about 1.1%, about 1.2%, about 1.3%, about 1.4%,about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%,about 2.1%, about 2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%,about 2.7%, about 2.8%, about 2.9%, about 3.0%, about 3.1%, about 3.2%,about 3.3%, about 3.4%, about 3.5%, about 3.6%, about 3.7%, about 3.8%,about 3.9%, about 4.0%, about 4.1%, about 4.2%, about 4.3%, about 4.4%,about 4.5%, about 4.6%, about 4.7%, about 4.8%, about 4.9%, about 5.0%,about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%,about 5.7%, about 5.8%, about 5.9%, about 6.0%, about 6.1%, about 6.2%,about 6.3%, about 6.4%, about 6.5%, about 6.6%, about 6.7%, about 6.8%,about 6.9%, about 7.0%, about 7.1%, about 7.2%, about 7.3%, about 7.4%,about 7.5%, about 7.6%, about 7.7%, about 7.8%, about 7.9%, about 8.0%,about 8.1%, about 8.2%, about 8.3%, about 8.4%, about 8.5%, about 8.6%,about 8.7%, about 8.8%, about 8.9%, about 9.0%, about 9.1%, about 9.2%,about 9.3%, about 9.4%, about 9.5%, about 9.6%, about 9.7%, about 9.8%,about 9.9%, about 10.0%, about 10.1%, about 10.2%, about 10.3%, about10.4%, about 10.5%, about 10.6%, about 10.7%, about 10.8%, about 10.9%,about 11.0%, about 11.1%, about 11.2%, about 11.3%, about 11.4%, about11.5%, about 11.6%, about 11.7%, about 11.8%, about 11.9%, about 12.0%,about 12.1%, about 12.2%, about 12.3%, about 12.4%, about 12.5%, about12.6%, about 12.7%, about 12.8%, about 12.9%, about 13.0%, about 13.1%,about 13.2%, about 13.3%, about 13.4%, about 13.5%, about 13.6%, about13.7%, about 13.8%, about 13.9%, about 14.0%, about 14.1%, about 14.2%,about 14.3%, about 14.4%, about 14.5%, about 14.6%, about 14.7%, about14.8%, about 14.9%, about 15.0%, about 15.1%, about 15.2%, about 15.3%,about 15.4%, about 15.5%, about 15.6%, about 15.7%, about 15.8%, about15.9%, about 16.0%, about 16.1%, about 16.2%, about 16.3%, about 16.4%,about 16.5%, about 16.6%, about 16.7%, about 16.8%, about 16.9%, about17.0%, about 17.1%, about 17.2%, about 17.3%, about 17.4%, about 17.5%,about 17.6%, about 17.7%, about 17.8%, about 17.9%, about 18.0%, about18.1%, about 18.2%, about 18.3%, about 18.4%, about 18.5%, about 18.6%,about 18.7%, about 18.8%, about 18.9%, about 19.0%, about 19.1%, about19.2%, about 19.3%, about 19.4%, about 19.5%, about 19.6%, about 19.7%,about 19.8%, about 19.9%, or about 20.0%.

In some embodiments, the tissue fillers of the invention includecross-linked SPF. In some embodiments, the tissue fillers of theinvention include cross-linked HA. An SPF fragment can be cross-linkedto another SPF fragment, or with HA. SPF-SPF, SPF-HA, and HA-HAcross-linked species can be obtained by using cross-linking agents ofvarious lengths, including zero length.

In some embodiments, the tissue fillers described herein may be providedin the form of a hydrogel having cross-linked HA and/or cross-linkedSPF. The cross-linked HA and/or cross-linked SPF (or SPF-HA cross-linkedspecies) may have a measurable degree of cross-linking. As used herein,the term “degree of crosslinking” refers to the number of cross-linkingunits (or molecules or residues) relative to the number of monomericunits in the polymer macromolecule, which was cross-linked. In someembodiments, the monomeric units are the amino acids in SPF. In someembodiments, the monomeric units are the disaccharide monomer units ofHA. Thus, a composition that that has a cross-linked matrix polymer witha 4% degree of crosslinking means that on average there are fourcrosslinking molecules for every 100 monomeric units. Every otherparameter being equal, the greater the degree of crosslinking, theharder the gel becomes. Without being limited to any one theory of theinvention, the degree of cross-linking in HA and/or SPF may result instiffer resulting materials or compositions prepared therefrom. Forexample, the higher the degree of cross-linking, the longer suchmaterials are likely to persist in the body. Indeed, without beinglimited to any one theory, biocompatible materials that includecross-linked materials will have varied rates of bioresorption,bioabsorption, and/or biodegradation depending on the degree ofcrosslinking where degree of cross-linking is inversely proportional tothe rate of bioresorption, bioabsorption, and/or biodegradation.Furthermore, greater crosslinking in the tissue fillers described hereinmay reduce hydrophilicity and the lifting capacity of such tissuefillers.

In a non-limiting example, a cross-linked SPF that has a degree ofcrosslinking of about 5%, has about 5 cross-linking moieties for every100 monomeric units, e.g., amino acids, in the cross-linked SPF.

Non-limiting examples of a degree of crosslinking include about 1% toabout 15%, or about 2% to about 14%, or about 1% to about 2%, about 1.5%to about 2.5%, or about 2% to about 3%, or about 2.5% to about 3.5%, orabout 3% to about 4%, or about 3.5% to about 4.5%, or about 4% to about5%, or about 4.5% to about 5.5%, or about 5% to about 6%, or about 5.5%to about 6.5%, or about 6% to about 7%, or about 6.5% or about 7.5%, orabout 7% to about 8%, or about 7.5% or about 8.5%, or about 8% to about9%, or about 8.5% to about 9.5%, or about 9% to about 10%, or about 9.5%to about 10.5%, or about 10% to about 11%, or about 10.5% to about11.5%, or about 11% to about 12%, or about 11.5% to about 12.5%, orabout 12% to about 13%, or about 12.5% to about 13.5%, or about 13% toabout 14%, or about 13.5% to about 14.5%, or about 14% to about 15%.

In some embodiments, the degree of crosslinking is at least 1%. In someembodiments, the degree of crosslinking is at least 2%. In someembodiments, the degree of crosslinking is at least 3%. In someembodiments, the degree of crosslinking is at least 4%. In someembodiments, the degree of crosslinking is at least 5%. In someembodiments, the degree of crosslinking is at least 6%. In someembodiments, the degree of crosslinking is at least 7%. In someembodiments, the degree of crosslinking is at least 8%. In someembodiments, the degree of crosslinking is at least 9%. In someembodiments, the degree of crosslinking is at least 10%. In someembodiments, the degree of crosslinking is at least 11%. In someembodiments, the degree of crosslinking is at least 12%. In someembodiments, the degree of crosslinking is at least 13%. In someembodiments, the degree of crosslinking is at least 14%. In someembodiments, the degree of crosslinking is at least 15%.

In some embodiments, a composition of the invention comprisescross-linked SPF where the degree of crosslinking is at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%,at least 13%, at least 14%, or at least 15%. In some embodiments, acomposition comprises cross-linked SPF where the degree of crosslinkingis at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, atmost 12%, at most 13%, at most 14%, or at most 15%. In some embodiments,a composition comprises cross-linked SPF where the degree ofcrosslinking is about 1% to about 15%, about 2% to about 11%, about 3%to about 10%, about 1% to about 5%, about 10% to about 15%, about 11% toabout 15%, about 6% to about 10%, or about 6% to about 8%, or about 1%to about 2%, about 1.5% to about 2.5%, or about 2^(%)to about 3%, orabout 2.5% to about 3.5%, or about 3% to about 4%, or about 3.5% toabout 4.5%, or about 4% to about 5%, or about 4.5% to about 5.5%, orabout 5% to about 6%, or about 5.5% to about 6.5%, or about 6% to about7%, or about 6.5% or about 7.5%, or about 7% to about 8%, or about 7.5%or about 8.5%, or about 8% to about 9%, or about 8.5% to about 9.5%, orabout 9% to about 10%, or about 9.5% to about 10.5%, or about 10% toabout 11%, or about 10.5% to about 11.5%, or about 11% to about 12%, orabout 11.5% to about 12.5%, or about 12% to about 13%, or about 12.5% toabout 13.5%, or about 13% to about 14%, or about 13.5% to about 14.5%,or about 14% to about 15%.

In some embodiments, a composition of the invention comprisescross-linked HA where the degree of crosslinking is at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%,at least 13%, at least 14%, or at least 15%. In some embodiments, acomposition comprises cross-linked HA where the degree of crosslinkingis at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, atmost 12%, at most 13%, at most 14%, or at most 15%. In some embodiments,a composition comprises cross-linked HA where the degree of crosslinkingis about 1% to about 15%, about 2% to about 11%, about 3% to about 10%,about 1% to about 5%, about 10% to about 15%, about 11% to about 15%,about 6% to about 10%, or about 6% to about 8%, or about 1% to about 2%,about 1.5% to about 2.5%, or about 2% to about 3%, or about 2.5% toabout 3.5%, or about 3% to about 4%, or about 3.5% to about 4.5%, orabout 4% to about 5%, or about 4.5% to about 5.5%, or about 5% to about6%, or about 5.5% to about 6.5%, or about 6% to about 7%, or about 6.5%or about 7.5%, or about 7% to about 8%, or about 7.5% or about 8.5%, orabout 8% to about 9%, or about 8.5% to about 9.5%, or about 9% to about10%, or about 9.5% to about 10.5%, or about 10% to about 11%, or about10.5% to about 11.5%, or about 11% to about 12%, or about 11.5% to about12.5%, or about 12% to about 13%, or about 12.5% to about 13.5%, orabout 13% to about 14%, or about 13.5% to about 14.5%, or about 14% toabout 15%.

In some embodiments, a composition of the invention comprisescross-linked SPF-HA where the degree of crosslinking is at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%,at least 13%, at least 14%, or at least 15%. In some embodiments, acomposition comprises cross-linked SPF-HA where the degree ofcrosslinking is at most 1%, at most 2, at most 3%, at most 4%, at most5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most11%, at most 12%, at most 13%, at most 14%, or at most 15%. In someembodiments, a composition comprises cross-linked SPF-HA where thedegree of crosslinking is about 1% to about 15%, about 2% to about 11%,about 3% to about 10%, about 1% to about 5%, about 10% to about 15%,about 11% to about 15%, about 6% to about 10%, or about 6% to about 8%,or about 1% to about 2%, about 1.5% to about 2.5%, or about 2% to about3%, or about 2.5% to about 3.5%, or about 3% to about 4%, or about 3.5%to about 4.5%, or about 4% to about 5%, or about 4.5% to about 5.5%, orabout 5% to about 6%, or about 5.5% to about 6.5%, or about 6% to about7%, or about 6.5% or about 7.5%, or about 7% to about 8%, or about 7.5%or about 8.5%, or about 8% to about 9%, or about 8.5% to about 9.5%, orabout 9% to about 10%, or about 9.5% to about 10.5%, or about 10% toabout 11%, or about 10.5% to about 11.5%, or about 11% to about 12%, orabout 11.5% to about 12.5%, or about 12% to about 13%, or about 12.5% toabout 13.5%, or about 13% to about 14%, or about 13.5% to about 14.5%,or about 14% to about 15%.

For example, 1 mole of SPF to 1 mole of HA may be cross linked whereinthe mole of HA could have a molecular weight of about 1 kDa to about 2 MkDa. In some embodiments, 1 mole of SPF to 1 million moles of HA, or visversa, where SPF can be 100 Da to 350 kDa, whereby any percentage ofeach mole can be cross-linked or free. A method of cross-linking SPF toother SPF can include one or more steps. In a first step, the epoxide,such as BDDE, is added to an SPF solution in excess and the reaction isallowed to proceed. Epoxides can react with various groups on the SPFmacromolecule, such as carboxyl, amine, alcohol, thiol, and the like,resulting in linkages such as esters, secondary or tertiary amines,ethers, thioethers, and the like. Where both epoxides of BDDE havereacted with the functional groups in one or more SPF macromolecules,the SPF becomes cross-linked. In an embodiment, cross-linking of HA maybe performed via a reaction with BDDE under alkaline conditions to yielda covalent linkage between HA and the cross-linker as described inSchanté et al., Carbohydrate Polymers (2011) 85:469-489. The degree ofmodification or crosslinking may be determined by NMR in accordance withmethods known in the art (e.g., Edsman et al., Dermatol. Surg. (2012)38: 1170-1179).

Methods of linking peptides are known in the art. The linking of theindividual isolated SPF into oligomeric and/or cross-linked SPF peptidesas set forth herein, can be effected by chemical conjugation procedureswell known in the art, such as by creating peptide linkages, use ofcondensation agents, and by employing well known bifunctionalcross-linking reagents. The conjugation may be direct, which includeslinkages not involving any intervening group, e.g., direct peptidelinkages, or indirect, wherein the linkage contains an interveningmoiety, such as a protein or peptide, e.g., plasma albumin, or otherspacer molecule. For example, the linkage may be via aheterobifunctional or homobifunctional cross-linker, e.g., carbodiimide,glutaraldehyde, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) andderivatives, bis-maleimide,4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the like.

Cross-linking can also be accomplished without exogenous cross-linkersby utilizing reactive groups on the molecules being conjugated. Methodsfor chemically cross-linking peptide molecules are generally known inthe art, and a number of hetero- and homobifunctional agents aredescribed in, e.g., U.S. Pat. Nos. 4,355,023, 4,657,853, 4,676,980,4,925,921, and 4,970,156, and Immuno Technology Catalogue and Handbook,Pierce Chemical Co. (1989), each of which is incorporated herein byreference. Such conjugation, including cross-linking, should beperformed so as not to substantially affect the desired function of thepeptide oligomer or entity conjugated thereto, including therapeuticagents, and moieties capable of binding substances of interest.

It will be understood to one skilled in the art that alternative linkerscan be used to link SPF peptides, for example the use of chemicalprotein cross-linkers. For example a homobifunctional cross-linker suchas disuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; 1,5,-2,4 dinitrobenzene orheterobifnictional cross-linkers such as N-hydroxysuccinimudyl2,3-dibromopropionate; 1-ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride; andsuccinimidyl-4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.

The present invention also provides compositions including cross-linkedSPF to HA. SPF to HA cross-linking can be achieved by various methods,for example by epoxide methods, periodate methods, and/or tresylchloride methods. In some embodiments, SPF are cross-linked to HA usingan epoxide, for example a multifunctional epoxide. For example, abifunctional epoxide such as 1,4 butanediol diglycidyl ether (BDDE) canbe used. Other multifunctional epoxides include, but are not limited to,polyglycerolpolyglycidyl ether (PGPGE), pentaerythriolpolyglycidyl ether(PEPGE) and diglycerolpolyglycidyl ether (DGPGE). Zero-lengthcross-linking between SPF and HA is also provided using an activatingagent.

A method of cross-linking SPF to other macromolecules, for example HA,can include one or more steps. In a first step, the epoxide, such asBDDE, is added to an SPF solution in excess and the reaction is allowedto proceed. Epoxides can react with various groups on the SPFmacromolecule, such as carboxyl, amine, alcohol, thiol, and the like,resulting in linkages such as esters, secondary or tertiary amines,ethers, thioethers, and the like. Where only one epoxide has reactedwith SPF, there remains a free epoxide attached to the SPF available forcross-linking with another SPF, or a different macromolecule, forexample HA, or the like. The order of adding the reagents can be varied.For example BDDE can be added to HA first, and then SPF is added to formcross-linked SPF-HA. In some embodiments, SPF and HA can be mixed first,and then BDDE is added to the mixture. In some embodiments, adding BDDEto a mixture of SPF and HA results in a composition includingcross-linked SPF to SPF, cross-linked HA to HA, and cross-linked SPF toHA.

In some embodiments, the cross-linked SPF-HA can be prepared using thetresyl chloride method, including one or more steps. In one step,cross-linked HA and/or non-cross-linked HA can be activated with tresylchloride, i.e., 2,2,2-trifluoroethanesulfonyl chloride, or any othersuitable acid chloride. Tresyl chloride is added for example drop-wiseto a base/solvent solution, for example, pyridine/acetone solution,containing cross-linked and/or non-cross-linked HA. In some embodiments,the tresyl chloride is reactive with all four of the hydroxyl groups onthe sugar rings of cross-linked and/or non-cross-linked HA. In anoptional step, the resulting HA-tresylate is washed. In a step, SPFfragments are added which will react with the HA-tresylate.

In some embodiments, the tresyl chloride method can be used to attach anSPF directly to cross-linked and/or non-cross-linked HA. In otherembodiments, the tresyl chloride method can be used to attach an SPF tocross-linked and/or non-cross-linked HA via a spacer, for example6-amino-1-hexanol. In some embodiments, the spacer can first be coupledto cross-linked or non-cross-linked HA via tresyl activation andcoupling. For coupling an SPF to the spacer, the tresyl activation andcoupling are thereafter repeated. Any suitable spacer can be used, i.e.,spacers having at least some characteristics similar to6-amino-1-hexanol, i.e., a primary amine for coupling to theHA-tresylate, and a reactive group, for example a hydroxyl group, foractivation and coupling of the SPF.

In some embodiments, tresyl chloride does not cross-link HA. The HAmatrix used in the tresyl chloride method may, however, be cross-linkedfor additional stability. The cross-linking can be effected, forexample, by using a multifunctional epoxide, such as BDDE, as describedabove. Cross-linking can be done either before or after peptidecoupling.

The tresyl chloride method has advantages over other immobilizationmethods, including efficient coupling under very mild conditions, noside reactions during activation and coupling, and the RGD peptides canbe bound directly to the carbon atoms of the HA support.

In various embodiments, tissue fillers described herein may include gelsand hydrogels that are HA-based. HA-based as used herein refers tocompositions or materials including cross-linked HA and compositionsincluding cross-linked HA plus one or more other cross-linked polymers.In addition, HA can refer to hyaluronic acid and any of its hyaluronatesalts, including, but not limited to, sodium hyaluronate (NaHA),potassium hyaluronate, magnesium hyaluronate, calcium hyaluronate, andcombinations thereof. The use of more than one biocompatible polymer isspecifically not excluded from the present description. Tissue fillersdescribed herein, which may be in the form gels and hydrogels, caninclude more than one biocompatible polymer, such as, for example, 2, 3,4, 5, 6, 7, 8, 9, 10, or more biocompatible polymers in addition to HAand/or SPF. Suitable biocompatible polymers include polysaccharides(e.g., HA, chitosan, chondroitin sulfate, alginate,carboxymethylcellulose), poly(ethyleneglycol), poly(lactic acid),poly(hydroxyethylmethacrylate), poly(methylmethacrylate), proteins otherthan SPF (e.g., elastin and collagen).

HA described herein may be intermolecularly cross-linked. In someembodiments, the cross-linking stabilizes HA physical properties. Insome embodiments, the present invention provides formation of stablecross-linked HA using multifunctional epoxides. As used herein, the term“multifunctional” epoxide means a chemical reagent having two or moreepoxides present, such as lower aliphatic epoxides or theircorresponding epihalohydrins. Examples of multifunctional epoxidesinclude, but are not limited to, the diepoxide 1,4 butanediol diglycidylether (BDDE), polyglycerolpolyglycidyl ether (PGPGE),pentaerythriolpolyglycidyl ether (PEPGE) and diglycerolpolyglycidylether (DGPGE). In a preferred embodiment, the diepoxide BDDE is used asthe cross-linking agent. The sugar moieties of HA cross-link via the twoepoxides of BDDE. In other embodiments, cross-linking agents includealkyldiepoxy bodies such as 1,3-butadiene diepoxide,1,2,7,8-diepoxyoctane, 1,5-hexadiene diepoxide and the like, diglycidylether bodies such as ethylene glycol diglycidyl ether, 1,4-butanedioldiglycidyl ether, bisphenol A diglycidyl ether and the like,divinylsulfone, and epichlorohydrin. Among them, particularly,divinylsulfone, 1,4-butanediol diglycidyl ether, and ethylene glycoldiglycidyl ether can be suitably used. In the present invention, two ormore kinds of crosslinking agents may be used by appropriately combiningthem.

In some embodiments, HA is cross-linked to HA. A method of cross-linkingHA to HA can include one or more steps. In a first step, an epoxide,such as BDDE, is added to an HA solution in excess and the reaction isallowed to proceed. Epoxides can react with from one to four of thehydroxyl groups on the sugar rings of HA to form one to four etherlinkages. Alternatively, or in addition to reacting with the hydroxylgroups, the epoxide can react with the carboxylic acid of thepolysaccharide to form an ester bond. Where both epoxides of BDDE havereacted with the fimctional groups in the sugar rings of one or more HAmacromolecules, the HA becomes cross-linked.

In some embodiments, the cross cross-linking agent can be a zero lengthcross-linking agent such as a chemical bond obtained by employing anactivating agent such as 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC), or BCDI. In some embodiments, the zero-length cross-linkingactivating agent is reacted with the HA in the presence ofN-hydroxysuccinimide (NHS), sulfo-NHS (or sulfonyl-NHS) or4-dimethylaminopyridine (DIMAP). In some embodiments, gels and hydrogelsdescribed herein are formed by reacting at least one cross-linkablebiocompatible polymer, such as HA and/or a protein, e.g. an SPF protein,or any other additional protein, with at least one cross-linkingactivating agent.

In some embodiments, cross-linked SPF-SPF, cross-linked SPF-HA, and/orcross-linked HA-HA, can have variable residence times after application,for example after being injected as an intra-dermal, subdermal, orgenerally, as a dermal filler. In some embodiments, residence times canbe affected in the sodium periodate method depending on the number ofreactive groups in the SPF which are available for attachment to anotherSPF macromolecule, or to HA. An example of a reactive group in SPF whichcan attach to HA is a primary amine. An SPF containing two reactivegroups, such as two primary amines, can itself cross-link the HA in theperiodate method, thereby creating a more stable conjugate. In otherembodiments, where only one reactive group is present in the SPF, suchas only one primary amine, for example at the amino terminus, SPF-HAcross-linking is reduced resulting in a more biodegradable matrix.

In some embodiments, BDDE cross-linked HA can have a variable residencetime after application, for example after being injected as anintra-dermal, subdermal, or generally dermal filler. In someembodiments, BDDE cross-linked HA can persist in dermal tissue anywherefrom one to at least thirty days, depending on the amount ofcross-linking. The variable residence time of the cross linked HA can betuned by introducing hydrolyzable bonds during the epoxidecross-linking. In some embodiments, the materials cross-linked withepoxide at a lower pH have a greater amount of ester bond formation andtherefore are more rapidly hydrolyzable.

In one embodiment, the cross-linking agent is a zero-lengthcross-linking activating agent. Generally, zero-length cross-linkingactivating agents couple polymers without adding any additional spacerarm atoms, and therefore zero-length cross-linking activating agents arenot incorporated into the cross-linked polymer matrix. Suitablezero-length cross-linking agents include carbodiimides, such as, forexample, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and BCDI.Non-water soluble carbodiimides include dicyclohexylcarbodiinide (DCC)and diisopropylcarbodiimide (DIC), which may also be suitable.

Carbodiimide-mediated coupling between carboxylates and alcohol or aminefunctional groups proceeds readily at ambient temperature, neutral pHand under aqueous conditions. Neutral pH can be, for example, betweenabout 6.0 and about 8.0, such as between about 6.5 and about 7.5, suchas about 7.0. Typically in water, l-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) can be used to mediate esterificationbetween carboxylates and alcohols or amidation between carboxylates andamines. Thus, cross-linked HA is formed by exploiting reactive groupspresent on HA (e.g., carboxylate and alcohol). In addition, by takingadvantage of the high reactivity of amine groups on proteins, forexample SPF proteins, amidation between lysine side-chains of proteinswith carboxylate groups of HA is achieved to form HA-proteincross-linked hydrogels. Cross-linking agents and unreacted polymers canbe removed by dialysis.

In some embodiments, EDC is used in conjunction withN-hydroxysuccinimide (NHS) or sulfonyl-NHS (sulfo-NHS), collectivelyreferred to as “NHS” herein. NHS stabilizes reactive intermediatesformed by EDC; thus, the addition of NHS can increase the couplingefficiency of EDC. Alternatively, 4-dimethylaminopyridine (DMAP) can beused to catalyze the coupling reaction.

In some embodiments, the HA-based tissue fillers of the inventioninclude cross-linked HA-based compositions and at least partiallycross-linked HA-based compositions. Uncross-linked HA as used hereinrefers to both truly uncross-linked (e.g., “free”) HA chains as well aslightly cross-linked chains and fragments thereof that are generally insoluble liquid form.

In some embodiments, the hydrogel compositions of the invention includesat least some cross-linking between HA and SPF.

Non-Limiting Exemplary Embodiments

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking occurring as a result of using an epoxy derivedcross-linker, e.g., BDDE, and with a degree of cross-linking of up to15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 5 kDa to about 150 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking occurring as a result of using an epoxy derivedcross-linker, e.g., BDDE, and with a degree of cross-linking of up to15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 6 kDa to about 17 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking occurring as a result of using an epoxy derivedcross-linker, e.g., BDDE, and with a degree of cross-linking of up to15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 17 kDa to about 39 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking occurring as a result of using an epoxy derivedcross-linker, e.g., BDDE, and with a degree of cross-linking of up to15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 39 kDa to about 80 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking occurring as a result of using an epoxy derivedcross-linker, e.g., BDDE, and with a degree of cross-linking of up to15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including low molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, hyaluronicacid (HA), water, and between about 0.05% to about 0.5% lidocaine, e.g.,about 0.3% lidocaine; wherein a portion of up to 100% w/w of SPF arecross-linked, and a portion of up to 100%, w/w of HA is cross linked,the cross-linking occurring between one or more of SPF to SPF, SPF toHA, and HA to HA; the cross-linking occurring as a result of using anepoxy derived cross-linker, e.g., BDDE, and with a degree ofcross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including medium molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, hyaluronicacid (HA), water, and between about 0.05% to about 0.5% lidocaine, e.g.,about 0.3% lidocaine; wherein a portion of up to 100% w/w of SPF arecross-linked, and a portion of up to 100% w/w of HA is cross linked, thecross-linking occumng between one or more of SPF to SPF, SPF to HA, andHA to HA; the cross-linking occurring as a result of using an epoxyderived cross-linker, e.g., BDDE, and with a degree of cross-linking ofup to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including low molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, mediummolecular weight silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, hyaluronic acid (HA), water, andbetween about 0.05% to about 0.5% lidocaine. e.g., about 0.3% lidocaine;wherein a portion of up to 100% w/w of SPF are cross-linked, and aportion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking occurring as a result of using an epoxy derivedcross-linker, e.g., BDDE, and with a degree of cross-linking of up to15%; wherein the w/w ratio between low molecular weight SPF and mediummolecular weight SPF is about 3:1.

In one embodiment, the invention relates to a biocompatible dermalfiller including high molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, hyaluronicacid (HA), water, and between about 0.05% to about 0.5% lidocaine, e.g.,about 0.3% lidocaine; wherein a portion of up to 100% w/w of SPF arecross-linked, and a portion of up to 100% w/w of HA is cross linked, thecross-linking occurring between one or more of SPF to SPF, SPF to HA,and HA to HA; the cross-linking occurring as a result of using an epoxyderived cross-linker, e.g., BDDE, and with a degree of cross-linking ofup to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 1 kDa to about 250 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking including zero-length cross-linking occurring as aresult of using an activating agent, e.g., BCDI, and with a degree ofcross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 5 kDa to about 150 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking including zero-length cross-linking occurring as aresult of using an activating agent, e.g., BCDI, and with a degree ofcross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 6 kDa to about 17 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine. e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking including zero-length cross-linking occurring as aresult of using an activating agent. e.g., BCDI, and with a degree ofcross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 17 kDa to about 39 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking including zero-length cross-linking occurring as aresult of using an activating agent. e.g., BCDI, and with a degree ofcross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0 and an average weight average molecularweight ranging from about 39 kDa to about 80 kDa, hyaluronic acid (HA),water, and between about 0.05% to about 0.5% lidocaine, e.g., about 0.3%lidocaine; wherein a portion of up to 100% w/w of SPF are cross-linked,and a portion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking including zero-length cross-linking occurring as aresult of using an activating agent. e.g., BCDI, and with a degree ofcross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including low molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, hyaluronicacid (HA), water, and between about 0.05% to about 0.5% lidocaine, e.g.,about 0.3% lidocaine; wherein a portion of up to 100% w/w of SPF arecross-linked, and a portion of up to 100% w/w of HA is cross linked, thecross-linking occurring between one or more of SPF to SPF, SPF to HA,and HA to HA; the cross-linking including zero-length cross-linkingoccurring as a result of using an activating agent, e.g., BCDI, and witha degree of cross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including medium molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, hyaluronicacid (HA), water, and between about 0.05% to about 0.5% lidocaine, e.g.,about 0.3% lidocaine; wherein a portion of up to 100% w/w of SPF arecross-linked, and a portion of up to 100% w/w of HA is cross linked, thecross-linking occurring between one or more of SPF to SPF, SPF to HA,and HA to HA; the cross-linking including zero-length cross-linkingoccurring as a result of using an activating agent, e.g., BCDI, and witha degree of cross-linking of up to 15%.

In one embodiment, the invention relates to a biocompatible dermalfiller including low molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, mediummolecular weight silk protein fragments (SPF) having a polydispersity ofbetween about 1.5 and about 3.0, hyaluronic acid (HA), water, andbetween about 0.05% to about 0.5% lidocaine, e.g., about 0.3% lidocaine;wherein a portion of up to 100% w/w of SPF are cross-linked, and aportion of up to 100% w/w of HA is cross linked, the cross-linkingoccurring between one or more of SPF to SPF, SPF to HA, and HA to HA;the cross-linking including zero-length cross-linking occurring as aresult of using an activating agent, e.g., BCDI, and with a degree ofcross-linking of up to 15%; wherein the w/w ratio between low molecularweight SPF and medium molecular weight SPF is about 3:1.

In one embodiment, the invention relates to a biocompatible dermalfiller including high molecular weight silk protein fragments (SPF)having a polydispersity of between about 1.5 and about 3.0, hyaluronicacid (HA), water, and between about 0.05% to about 0.5% lidocaine, e.g.,about 0.3% lidocaine; wherein a portion of up to 100% w/w of SPF arecross-linked, and a portion of up to 100% w/w of HA is cross linked, thecross-linking occurring between one or more of SPF to SPF, SPF to HA,and HA to HA; the cross-linking including zero-length cross-linkingoccurring as a result of using an activating agent, e.g., BCDI, and witha degree of cross-linking of up to 15%.

Additional Agents

In some embodiments, the tissue fillers described herein include anactive agent, such as a drug. In some embodiments, the active agent canbe one or more of enzyme inhibitors, anesthetic agents, medicinalneurotoxins, antioxidants, anti-infective agents, anti-inflammatoryagents, vasodilators, ultraviolet (UV) light blocking agents, dyes(e.g., tattoo dye, ink or pigment), a reflective agent, hormones,immunosuppressants, and combinations thereof. The tissue fillersdescribed herein can include an active agent selected from the groupconsisting of enzyme inhibitors, anesthetic agents, medicinalneurotoxins (e.g., botulinum toxin and clostridium toxin), antioxidants,anti-infective agents (e.g., antibiotics), vasodilators, dyes (e.g.,tattoo ink or pigment, reflective agents, anti-inflammatory agents,ultraviolet (UV) light blocking agents, dyes, hormones,immunosuppressants, and combinations thereof.

In some embodiments, the immunosuppressant is rapamycin, orrapamycin-like compound.

In some embodiments, the active agent may be an antibiotic selected fromthe group consisting of a penicillin (e.g., penicillin V, amoxicillin),an erythromycin (e.g., erythromycin stearate), a lincosamide (e.g.,clindamycin), and a cephalosporin (e.g. cephalexin), and a combinationthereof.

In some embodiments, the active agent may be a vasodilator selected fromthe group consisting of nitroglycerin, labetalol, thrazide, isosorbidedinitrate, pentaerythritol tetranitrate, digitalis, hydralazine,diazoxide, amrinone, L-arginine, bamethan sulphate, bencyclane fumarate,benfurodil hemisuccinate, benzyl nicotinate, buflomedil hydrochloride,buphenine hydrochloride, butalamine hydrochloride, cetiedil citrate,ciclonicate, cinepazide maleate, cyclandelate, di-isopropylammoniumdichloroacetate, ethyl nicotinate, hepronicate, hexyl nicotinate,ifenprodil tartrate, inositol nicotinate, isoxsuprine hydrochloride,kallidinogenase, methyl nicotinate, naftidrofuyl oxalate, nicametatecitrate, niceritrol, nicoboxil, nicofuranose, nicotinyl alcohol,nicotinyl alcohol tartrate, nitric oxide, nonivamide, oxpentifylline,papaverine, papaveroline, pentifylline, peroxynitrite, pinacidil,pipratecol, propentofyltine, raubasine, suloctidil, teasuprine,thymoxamine hydrochloride, tocopherol nicotinate, tolazoline, xanthinolnicotinate, diazoxide, hydralazine, minoxidil, and sodium nitroprusside,and a combination thereof.

In some embodiments, the tissue fillers described herein may include anactive agent at a concentration, by weight, of at least 0.01%, or atleast 0.02%, or at least 0.03%, or at least 0.04%, or at least 0.05%, orat least 0.06%, or at least 0.07%, or at least 0.08%, or at least 0.09%,or at least 0.1%, or at least 0.2%, or at least 0.3%, or at least 0.4%,or at least 0.5%, or at least 0.6%, or at least 0.7%, or at least 0.8%,or at least 0.9%, or at least 1.0%, or at least 1.5%, or at least 2.0%,or at least 2.5%, or at least 3.0%, or at least 3.5%, or at least 4.0%,or at least 4.5%, or at least 5.0%, or at least 5.5%, or at least 6.0%,or at least 6.5%, or at least 7.0%, or at least 7.5%, or at least 8.0%,or at least 8.5%, or at least 9.0%, or at least 9.5%, or at least 10%,or at least 15%, or at least 20%, or at least 25%, or at least 30%, orat least 35%, or at least 40%, or at least 45%, or at least 50%.

In some embodiments, the tissue fillers described herein may include anactive agent at a concentration, by weight, of at most 0.01%, or at most0.02%, or at most 0.03%, or at most 0.04%, or at most 0.05%, or at most0.06%, or at most 0.07%, or at most 0.08%, or at most 0.09%, or at most0.1%, or at most 0.2%, or at most 0.3%, or at most 0.4%, or at most0.5%, or at most 0.6%, or at most 0.7%, or at most 0.8%, or at most0.9%, or at most 1.0%, or at most 1.5%, or at most 2.0%, or at most2.5%, or at most 3.0%, or at most 3.5%, or at most 4.00%, or at most4.5%, or at most 5.0%, or at most 5.5%, or at most 6.0%, or at most6.5%, or at most 7.0%, or at most 7.5%, or at most 8.0%, or at most8.5%, or at most 9.0%, or at most 9.5%, or at most 10%, or at most 15%,or at most 20%, or at most 25%, or at most 30%, or at most 35%, or atmost 40%, or at most 45%, or at most 50%.

In some embodiments, the tissue fillers described herein may include anactive agent at a concentration, by weight, of about 0.01% to about0.1%, or about 0.05% to about 0.15%, or about 0.1% to about 0.2%, orabout 0.15% to about 0.25%, or about 0.2% to about 0.3%, or about 0.25%to about 0.35%, or about 0.3% to about 0.4%, or about 0.35% to about0.45%, or about 0.4% to about 0.5%, or about 0.45% to about 0.55%, orabout 0.5% to about 0.6%, or about 0.55% to about 0.65%, or about 0.6%to about 0.7%, or about 0.65% to about 0.75%, or about 0.7% to about0.8%, or about 0.75% to about 0.85%, or about 0.8% to about 0.9%, orabout 0.85% to about 0.95%, or about 1% to about 2%, or about 1.5% toabout 2.5%, or about 2% to about 3%, or about 2.5% to about 3.5%, orabout 3% to about 4%, or about 3.5% to about 4.5%, or about 4% to about5%, or about 4.5% to about 5.5%, or about 5% to about 6%, or about 5.5%to about 6.5%, or about 6% to about 7%, or about 6.5% to about 7.5%, orabout 7% to about 8%, or about 7.5% to about 8.5%, or about 8% to about9%, or about 8.5% to about 9.5%, or about 9% to about 10%, or about 10%to about 15%, or about 15% to about 20%, or about 20% to about 25%, orabout 25% to about 30%, or about 30% to about 35%, or about 35% to about40%, or about 40% to about 45%, or about 45% to about 50%.

In some embodiments, the tissue fillers described herein may include anactive agent at a concentration, by weight, of about 0.01%, or about0.02%, or about 0.03%, or about 0.04%, or about 0.05%, or about 0.06%,or about 0.07%, or about 0.08%, or about 0.09%, or about 0.1%, or about0.2%, or about 0.3%, or about 0.4%, or about 0.5%, or about 0.6%, orabout 0.7%, or about 0.8%, or about 0.9%, or about 1.0%, or about 1.5%,or about 2.0%, or about 2.5%, or about 3.0%, or about 3.5%, or about4.0%, or about 4.5%, or about 5.0%, or about 5.5%, or about 6.0%, orabout 6.5%, or about 7.0%, or about 7.5%, or about 8.0%, or about 8.5%,or about 9.0%, or about 9.5%, or about 10%, or about 11%, or about 12%,or about 13%, or about 14%, or about 15%, or about 16%, or about 17%, orabout 18%, or about 19%, or about 20%, or about 21%, or about 22%, orabout 23%, or about 24%, or about 25%, or about 26%, or about 27%, orabout 28%, or about 29%, or about 30%, or about 31%, or about 32%, orabout 33%, or about 34%, or about 35%, or about 36%, or about 37%, orabout 38%, or about 39%, or about 40%, or about 41%, or about 42%, orabout 43%, or about 44%, or about 45%, or about 46%, or about 47%, orabout 48%, or about 49%, or about 50%.

In some embodiments, the tissue fillers described herein include afibrosis-inhibiting agent. In some embodiments, tissue fillers describedherein may further include a compound that acts to have an inhibitoryeffect on pathological processes in or around the treatment site. Incertain aspects, the active agent may be selected from one of thefollowing classes of compounds: anti-inflammatory agents (e.g.,dexamethasone, cortisone, fludrocortisone, prednisone, prednisolone,6α-methylprednisolone, triamcinolone, betamethasone, and aspirin).

In some embodiments, with the active agent may, but is not limited to,antioxidants and enzymes. In an embodiment, the active agent mayinclude, but is not limited to, selenium, ubiquinone derivatives,thiol-based antioxidants, saccharide-containing antioxidants,polyphenols, botanical extracts, caffeic acid, apigenin, pycnogenol,resveratrol, folic acid, vitamin B12, vitamin B6, vitamin B3, vitamin E,vitamin C and derivatives thereof, vitamin D, vitamin A, astaxathin,lutein, lycopene, essential fatty acids (omegas 3 and 6), iron, zinc,magnesium, flavonoids (soy, curcumin, silymarin, pycnongeol), growthfactors, aloe, hyaluronic acid, extracellular matrix proteins, cells,nucleic acids, biomarkers, biological reagents, zinc oxide, benzoylperoxide, retinoids, titanium, allergens in a known dose (forsensitization treatment), essential oils including, but not limited to,lemongrass or rosemary oil, and fragrances. Considering the activeagents more broadly, the active agents may include therapeutic agentssuch as small molecules, drugs, proteins, peptides and nucleic acids.

In certain embodiments, the tissue fillers described herein can includeone or more anesthetic agents in an amount effective to ameliorate ormitigate pain or discomfort at the tissue filler injection site. Thelocal anesthetic can be selected from the group of ambucaine, amolanone,amylocalne, benoxinate, benzocaine, betoxycaine, biphenamine,bupivacaine, butacaine, butamben, butanilicaine, butethamine,butoxycaine, carticaine, chloroprocaine, cocaethylene, cocaine,cyclomethycaine, dibucaine, dimethisoquin, dimethocaine, diperodon,dicyclomine, ecgonidine, ecgonine, ethyl chloride, etidocaine,beta-eucaine, euprocin, fenalcomine, formocaine, hexylcaine,hydroxytetracaine, isobutyl p-aminobenzoate, leucinocaine mesylate,levoxadrol, lidocaine, mepivacaine, meprylcaine, metabutoxycaine, methylchloride, myrtecaine, naepaine, octacaine, orthocaine, oxethazaine,parethoxycaine, phenacaine, phenol, piperocaine, piridocaine,polidocanol, pramoxine, prilocalne, procaine, propanocaine,proparacaine, propipocaine, propoxycaine, pseudococaine, pyrrocaine,ropivacaine, salicyl alcohol, tetracaine, tolycaine, trimecaine,zolamine, and salts thereof.

In some embodiments, the tissue fillers described herein may includelidocaine or other anesthetic recited above at a concentration, byweight, of at least 0.01%, or at least 0.02%, or at least 0.03%, or atleast 0.04%, or at least 0.05%, or at least 0.06%, or at least 0.07%, orat least 0.08%, or at least 0.09%, or at least 0.1%, or at least 0.2%,or at least 0.3%, or at least 0.4%, or at least 0.5%, or at least 0.6%,or at least 0.7%, or at least 0.8%, or at least 0.9%, or at least 1.0%,or at least 1.5%, or at least 2.0%, or at least 2.5%, or at least 3.0%,or at least 3.5%, or at least 4.0%, or at least 4.5%, or at least 5.0%,or at least 5.5%, or at least 6.0%, or at least 6.5%, or at least 7.0%,or at least 7.5%, or at least 8.0%, or at least 8.5%, or at least 9.0%,or at least 9.5%, or at least 10%.

In some embodiments, the tissue fillers described herein may includelidocaine or other anesthetic recited above at a concentration, byweight, of at most 0.01%, or at most 0.02%, or at most 0.03%, or at most0.04%, or at most 0.05%, or at most 0.06%, or at most 0.07%, or at most0.08%, or at most 0.09%, or at most 0.1%, or at most 0.2%, or at most0.3%, or at most 0.4%, or at most 0.5%, or at most 0.6%, or at most0.7%, or at most 0.8%, or at most 0.9%, or at most 1.0%, or at most1.5%, or at most 2.0, or at most 2.5%, or at most 3.0%, or at most 3.5%,or at most 4.0%, or at most 4.5%, or at most 5.0%, or at most 5.5%, orat most 6.0%, or at most 6.5%, or at most 7.0%, or at most 7.5%, or atmost 8.0%, or at most 8.5%, or at most 9.0%, or at most 9.5%, or at most10%.

In some embodiments, the tissue fillers described herein may includelidocaine or other anesthetic recited above at a concentration, byweight, of about 0.01%, or about 0.02%, or about 0.03%, or about 0.04%,or about 0.05%, or about 0.06%, or about 0.07%, or about 0.08%, or about0.09%, or about 0.1%, or about 0.2%, or about 0.3%, or about 0.4%, orabout 0.5%, or about 0.6%, or about 0.7%, or about 0.8%, or about 0.9%,or about 1.0%, or about 1.5%, or about 2.0%, or about 2.5%, or about3.0%, or about 3.5%, or about 4.0%, or about 4.5%, or about 5.0%, orabout 5.5%, or about 6.0%, or about 6.5%, or about 7.0%, or about 7.5%,or about 8.0%, or about 8.5%, or about 9.0%, or about 9.5%, or about10%.

In some embodiments, the tissue fillers described herein may includelidocaine or other anesthetic recited above at a concentration, byweight, of about 0.01% to about 0.02%, or about 0.03% to about 0.04%, orabout 0.05% to about 0.06% to about 0.07%, or about 0.08% to about0.09%, or about 0.1% to about 0.2%, or about 0.3% to about 0.4%, orabout 0.5% to about 0.6%, or about 0.7% to about 0.8%, or about 0.9% toabout 1.0%, or about 1% to about 1.5%, or about 1.5% to about 2.0%, orabout 2.0% to about 2.5%, or about 2.5% to about 3.0%, or about 3.0% toabout 3.5%, or about 3.5% to about 4.0%, or about 4.0% to about 4.5%, orabout 4.5% to about 5.0%, or about 5.0% to about 5.5%, or about 5.5% toabout 6.0%, or about 6.0% to about 6.5%, or about 6.5% to about 7.0%, orabout 7.5% to about 8.0%, or about 8.0% to about 8.5%, or about 8.5% toabout 9.0%, or about 9.5% to about 10%.

In one embodiment, the anesthetic agent is lidocaine, such as in theform of lidocaine HCl. The tissue fillers described herein may have alidocaine or other anesthetic in a concentration of between about 0.1%and about 5% by weight of the composition, for example, about 0.2% toabout 1.0% by weight of the tissue filler. In one embodiment, the tissuefiller has a lidocaine concentration of about 0.3% by weight (w/w %) ofthe tissue filler. The concentration of lidocaine in the tissue fillersdescribed herein can be therapeutically effective meaning theconcentration is adequate to provide a therapeutic benefit such as, forexample, ameliorating or mitigating pain or discomfort at the tissuefiller injection site.

Optical Properties

When light encounters a material, it can interact with it in severalways. These interactions depend on the nature of the light, i.e., itswavelength, frequency, energy, etc., and the nature of the material.Light interacts with an object by some combination of reflection, andtransmittance with refraction. An optically transparent material allowsmuch of the light that falls on it to be transmitted, with little lightbeing reflected. Materials which do not allow the transmission of lightare called optically opaque, or simply opaque.

In some embodiments, the invention provides a tissue filler describedherein having transparency and/or translucency. Transparency (alsocalled pellucidity or diaphaneity) is the physical property of allowinglight to pass through a material, whereas translucency (also calledtranslucence or translucidity) only allows light to pass throughdiffusely. The opposite property is opacity. Transparent materials areclear, while translucent ones cannot be seen through clearly. The tissuefillers disclosed herein may, or may not, exhibit optical propertiessuch as transparency and/or translucency. In some embodiments, includingmethods for superficial line filling, it would be an advantage to havean opaque hydrogel. Factors used to control a tissue filler's opticalproperties include, without limitation, SPF concentration, degree ofcrystallinity, and/or hydrogel homogeneity.

In some embodiments, the tissue fillers described herein are opaque.

In an embodiment, a tissue filler described herein is opticallytransparent. In aspects of this embodiment, a tissue filler describedherein transmits, e.g., about 75% of the light, about 80% of the light,about 85% of the light, about 90% of the light, about 95% of the light,or about 100% of the light. In other aspects of this embodiment, atissue filler described herein, e.g., at least 75% of the light, atleast 80% of the light, at least 85% of the light, at least 90% of thelight, or at least 95% of the light. In yet other aspects of thisembodiment, an a tissue filler described herein transmits, e.g., about75% to about 100% of the light, about 80% to about 100% of the light,about 85% to about 100° % of the light, about 90% to about 100% of thelight, or about 95% to about 100% of the light.

In another embodiment, a tissue filler described herein is opticallyopaque. In aspects of this embodiment, a tissue filler described hereintransmits, e.g., about 0.1% of the light, about 1% of the light, about10% of the light, about 15% of the light, about 20% of the light, about25% of the light, about 30% of the light, about 35% of the light, about40% of the light, about 45% of the light, about 50% of the light, about55% of the light, about 60% of the light, about 65% of the light, about70% of the light, about 75% of the light, about 80% of the light, about85% of the light, about 90% of the light, about 95% of the light, orabout 100% of the light. In other aspects of this embodiment, a tissuefiller described herein transmits, e.g., at most 0.1% of the light, atmost 1% of the light, at most 10% of the light, at most 15% of thelight, at most 20% of the light, at most 25% of the light, at most 30%of the light, at most 35% of the light, at most 40% of the light, atmost 45% of the light, at most 50% of the light, at most 55% of thelight, at most 60% of the light, at most 65% of the light, at most 70%of the light, or at most 75% of the light. In other aspects of thisembodiment, a tissue filler described herein transmits, e.g., at least0.1% of the light, at least 1% of the light, at least 10% of the light,at least 15% of the light, at least 20% of the light, at least 25% ofthe light, at least 30% of the light, at least 35% of the light, atleast 40% of the light, at least 45% of the light, at least 50% of thelight, at least 55% of the light, at least 60% of the light, at least65% of the light, at least 70% of the light, or at least 75% of thelight. In other aspects of this embodiment, a tissue filler describedherein transmits, e.g., about 0.1% to about 15%, about 0.1% to about20%, about 0.1% to about 25%, about 0.1% to about 30%, about 0.1% toabout 35%, about 0.1% to about 40%, about 0.1% to about 45%, about 0.1%to about 50%, about 0.1% to about 55%, about 0.1% to about 60%, about0.1% to about 65%, about 0.1% to about 70%, about 0.1% to about 75%,about 1% to about 15%, about 1% to about 20%, about 1% to about 25%,about 1% to about 30%, about 1% to about 35%, about 1% to about 40%,about 1% to about 45%, about 1% to about 50%, about 1% to about 55%,about 1% to about 60%, about 1% to about 65%, about 1% to about 70%/o,about 1% to about 75%, about 10% to about 20%, about 10% to about 25%,about 10% to about 30%, about 10% to about 35%, about 10% to about 40%,about 10% to about 45%, about 10% to about 50%, about 10% to about 55%,about 10% to about 60%, about 10% to about 65%, about 10% to about 70%,about 10% to about 75%, about 25% to about 35%, about 25% to about 40%,about 25% to about 45%, about 25% to about 50%, about 25% to about 55%,about 25% to about 60%, about 25% to about 65%, about 25% to about 70%,or about 25% to about 75%, of the light.

In some embodiments, a tissue filler described herein is opticallytranslucent. In aspects of this embodiments, a tissue filler describedherein diffusely transmits, e.g., about 75% of the light, about 80% ofthe light, about 85% of the light, about 90% of the light, about 95% ofthe light, or about 100% of the light. In other aspects of theseembodiments, a tissue filler diffusely transmits, e.g., at least 0.1% ofthe light, at least 1% of the light, at least 5% of the light, at least10% of the light, at least 15% of the light, at least 20% of the light,at least 25% of the light, at least 30% of the light, at least 35% ofthe light, at least 40% of the light, at least 45% of the light, atleast 50% of the light, at least 55% of the light, at least 60% of thelight, at least 65% of the light, at least 70% of the light, 75% of thelight, at least 80% of the light, at least 85% of the light, at least90% of the light, or at least 95% of the light. In other aspects ofthese embodiments, a tissue filler diffusely transmits, e.g., at most0.1% of the light, at most 1% of the light, at most 5% of the light, atmost 10% of the light, at most 15% of the light, at most 20% of thelight, at most 25% of the light, at most 30% of the light, at most 35%of the light, at most 40% of the light, at most 45% of the light, atmost 50% of the light, at most 55% of the light, at most 60% of thelight, at most 65% of the light, at most 70% of the light, 75% of thelight, at most 80% of the light, at most 85% of the light, at most 90%of the light, at most 95% of the light, or at most 100% of the light. Inyet other aspects of these embodiments, a tissue filler diffuselytransmits, e.g., about 0.1% to about 100% of the light, about 1% toabout 100% of the light, about 5% to about 100% of the light, about 10%to about 100% of the light, about 15% to about 100% of the light, about20% to about 100% of the light, about 25% to about 100% of the light,about 30% to about 100% of the light, about 35% to about 100% of thelight, about 45% to about 100% of the light, about 50% to about 100% ofthe light, about 55% to about 100% of the light, about 60% to about 100%of the light, about 65% to about 100% of the light, about 70% to about100% of the light, about 75% to about 100% of the light, about 80% toabout 100% of the light, about 85% to about 100% of the light, about 90%to about 100% of the light, or about 95% to about 100% of the light.

In some embodiments, a tissue filler described herein may be describedby its attenuation coefficient, which is defined as a description ofmaterial's ability to scatter or absorb light.

Tissue filler and skin properties can influence the manifestation of theadverse Tyndall effect event in skin following delivery of certaintissue fillers known in the art. Fillers with high stiffness andelasticity can be used to correct areas on the face like nasolabialfolds, cheeks, and chin without any fear of facial discoloration, as thematerials are injected in the mid and deep dermis regions. However, whenfillers are used for more superficial applications, for example tocorrect fine line wrinkles, or mistakenly applied too superficially inthe upper regions of the dermis, a bluish discoloration of the skin isoften observed. This phenomenon, which is thought to be the result ofTyndall effect, leaves a semi-permanent discoloration of the applicationsites. In some embodiments, the effect disappears after theadministration of enzymes, for example hyaluronidase, in order todegrade the filler material. Consequently, Tyndall effect is more commonin patients treated for superficial fine line wrinkles. Prolongedmanifestation of Tyndall effect, typically for as long as the fillerlasts in the skin, is an undesired side effect and a cause of concernfor patients.

In some embodiments, the tissue fillers described herein mitigate theTyndall effect due to their homogeneity and resulting opacity.

In some embodiments, the tissue fillers described herein do not resultin Tyndall effect, or do not result in any visually perceptible bluediscoloration resulting from Tyndall effect. In some embodiments, theinvention relates to tissue fillers and methods for improving aestheticappearance, comprising administering, to a dermal region of a patient, asubstantially optically transparent dermal filler composition thatexhibits no or insignificant Tyndall effect. The appearance of a bluediscoloration at the skin site where a tissue filler had been injected,(Tyndall effect) is a significant adverse event experienced by somedermal filler patients. Tyndall effect is more common in patientstreated for superficial fine line wrinkles. Embodiments of the presentinvention have been developed which provide long lasting, translucentfillers which can be injected superficially to treat fine lines andwrinkles, even in regions of relatively thin skin, without any resultingblue discoloration from Tyndall effect. Fine lines or superficialwrinkles are generally understood to be those wrinkles or creases inskin that are typically found in regions of the face (forehead, lateralcanthus, vermillion border/perioral lines) where the skin is thinnest,that is, the skin has a dermis thickness of less than 1 mm. On theforehead the average dermal thickness is about 0.95 mm for normal skinand about 0.81 mm for wrinkled skin. Dermis around the lateral canthusis even thinner (e.g., about 0.61 mm for normal skin and about 0.41 mmfor wrinkled skin). The average outer diameter of a 30 or 32 gaugeneedle (needles that are typically used for fine line gel application)is about 0.30 and about 0.24 mm.

In an embodiment, a tissue filler disclosed herein is optically opaque.In aspects of this embodiment, a tissue filler disclosed hereintransmits. e.g., about 5% of the light, about 10% of the light, about15% of the light, about 20% of the light, about 25% of the light, about30% of the light, about 35% of the light, about 40% of the light, about45% of the light, about 50% of the light, about 55% of the light, about60% of the light, about 65% of the light, or about 70% of the light. Inother aspects of this embodiment, a tissue filler disclosed hereintransmits, e.g., at most 5% of the light, at most 10% of the light, atmost 15% of the light, at most 20% of the light, at most 25% of thelight, at most 30% of the light, at most 35% of the light, at most 40%of the light, at most 45% of the light, at most 50% of the light, atmost 55% of the light, at most 60% of the light, at most 65% of thelight, at most 70% of the light, or at most 75% of the light. In otheraspects of this embodiment, a tissue filler disclosed herein transmits,e.g., about 5% to about 15%, about 5% to about 20%, about 5% to about25%, about 5% to about 30%, about 5% to about 35%, about 5% to about40%, about 5% to about 45%, about 5% to about 50%, about 5% to about55%, about 5% to about 60%, about 5% to about 65%, about 5% to about70%, about 5% to about 75%, about 15% to about 20%, about 15% to about25%, about 15% to about 30%, about 15% to about 35%, about 15% to about40%, about 15% to about 45%, about 15% to about 50%, about 15% to about55%, about 15% to about 60%, about 15% to about 65%, about 15% to about70%, about 15% to about 75%, about 25% to about 35%, about 25% to about40%, about 25% to about 45%, about 25% to about 50%, about 25% to about55%, about 25% to about 60%, about 25% to about 65%, about 25% to about70%, or about 25% to about 75%, of the light.

In some embodiments, a tissue filler disclosed herein exhibits, e.g.,about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about100% reduction in tyndalling. In other aspects of these embodiments, atissue filler disclosed herein exhibits, e.g., at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least35%, at least 40%, at least 45%, at least 50%, at least 55%, at least60%, at least 65%, at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, or at least 100%, reduction intyndalling. In other aspects of these embodiments, a tissue fillerdisclosed herein exhibits, e.g., about 20% to about 100%, about 50% toabout 100%, about 70% to about 100%, about 15% to about 35%, about 20%to about 40%, about 25% to about 45%, about 30% to about 50%, about 35%to about 55%, about 40% to about 60%, about 45% to about 65%, about 50%to about 70%, about 55% to about 75%, about 60% to about 80%, about 65%to about 85%, about 70% to about 90%, about 75% to about 95%, or about80% to about 100%, reduction in tyndalling.

water Content

In an embodiment, the tissue fillers described herein may include water.For example, some tissue fillers described herein may be gels, such ashydrogels, and may include water absorbed, entrapped, or otherwisedisposed therein.

In an embodiment, the percent water content, by weight, in the tissuefillers of the present disclosure is 1% to 95%. In an embodiment, thepercent water content, by weight, in the tissue fillers described hereinis at least 1%, or at least 2%, or at least 3%, or at least 4%, or atleast 5%, or at least 6%, or at least 7%, or at least 8%, or at least9%, or at least 10%, or at least 11%, or at least 12%, or at least 13%,or at least 14%, or at least 15%, or at least 16%, or at least 17%, orat least 18%, or at least 19%, or at least 20%, or at least 21%, or atleast 22%, or at least 23%, or at least 24%, or at least 25%, or atleast 26%, or at least 27%, or at least 28%, or at least 29%, or atleast 30%, or at least 31%, or at least 32%, or at least 33%, or atleast 34%, or at least 35%, or at least 36%, or at least 37%, or atleast 38%, or at least 39%, or at least 40%, or at least 41%, or atleast 42%, or at least 43%, or at least 44%, or at least 45%, or atleast 46%, or at least 47%, or at least 48%, or at least 49%, or atleast 50%, or at least 51%, or at least 52%, or at least 53%, or atleast 54%, or at least 55%, or at least 56%, or at least 57%, or atleast 58%, or at least 59%, or at least 60%, or at least 61%, or atleast 62%, or at least 63%, or at least 64%, or at least 65%, or atleast 66%, or at least 67%, or at least 68%, or at least 69%, or atleast 70%, or at least 71%, or at least 72%, or at least 73%, or atleast 74%, or at least 75%, or at least 76%, or at least 77%, or atleast 78%, or at least 79%, or at least 80%, or at least 81%, or atleast 82%, or at least 83%, or at least 84%, or at least 85%, or atleast 86%, or at least 87%, or at least 88%, or at least 89%, or atleast 90%, or at least 91%, or at least 92%, or at least 93%, or atleast 94%, or at least 95%.

In an embodiment, the percent water content, by weight, in the tissuefillers described herein is at most 1%, or at most 2%, or at most 3%, orat most 4%, or at most 5%, or at most 6%, or at most 7%, or at most 8%,or at most 9%, or at most 10%, or at most 11%, or at most 12%, or atmost 13%, or at most 14%, or at most 15%, or at most 16%, or at most17%, or at most 18%, or at most 19%, or at most 20%, or at most 21%, orat most 22%, or at most 23%, or at most 24%, or at most 25%, or at most26%, or at most 27%, or at most 28%, or at most 29%, or at most 30%, orat most 31%, or at most 32%, or at most 33%, or at most 34%, or at most35%, or at most 36%, or at most 37%, or at most 38%, or at most 39%, orat most 40%, or at most 41%, or at most 42%, or at most 43%, or at most44%, or at most 45%, or at most 46%, or at most 47%, or at most 48%, orat most 49%, or at most 50%, or at most 51%, or at most 52%, or at most53%, or at most 54%, or at most 55%, or at most 56%, or at most 57%, orat most 58%, or at most 59%, or at most 60%, or at most 61%, or at most62%, or at most 63%, or at most 64%, or at most 65%, or at most 66%, orat most 67%, or at most 68%, or at most 69%, or at most 700%, or at most71%, or at most 72%, or at most 73%, or at most 74%, or at most 75%, orat most 76%, or at most 77%, or at most 78%, or at most 79%, or at most80%, or at most 81%, or at most 82%, or at most 83%, or at most 84%, orat most 85%, or at most 86%, or at most 87%, or at most 88%, or at most89%, or at most 90%, or at most 91%, or at most 92%, or at most 93%, orat most 94%, or at most 95%.

In an embodiment, the percent water content, by weight, in the tissuefillers described herein is 1% to 2%, or 2% to 3%, or 3% to 4%, or 4% to5%, or 5% to 6%, or 6% to 7%, or 7% to 8%, or 8% to 9%, or 9% to 10%, or10% to 11%, or 11% to 12%, or 12% to 13%, or 13% to 14%, or 14% to 15%,or 15% to 16%, or 16% or 17%, or 17% to 18%, or 18% to 19%, or 19% to20%, or 20% to 21%, or 21% to 22%, or 22% to 23%, or 23% to 24%, or 24%to 25%, or 25% to 26%, or 26% to 27%, or 27% to 28%, or 28% to 29%, or30% to 31%, or 31% to 32%, or 32% to 33%, or 33% to 34%, or 34% to 35%,or 35% to 36%, or 36% to 37%, or 37% to 38%, or 38% to 39%, or 39% to40%, or 40% to 41%, or 41% to 42%, or 42% to 43%, or 43% to 44%, or 44%to 45%, or 45% to 46%, or 46% to 47%, or 47% to 48%, or 48% to 49%, or49% to 500%, or 50% to 51%, or 51% to 52%, or 52% to 53%, or 53% to 54%,or 54% to 55%, or 55% to 56%, or 56% to 57%, or 57% to 58%, or 58% to59%, or 59% to 60%, or 60% to 61%, or 61% to 62%, or 62% to 63%, or 63%to 64%, or 64% to 65%, or 65% to 66%, or 66% to 67%, or 67% to 68%, or68% to 69%, or 69% to 70%, or 70% to 71%, or 71% to 72%, or 72% to 73%,or 73% to 74%, or 74% to 75%, or 75% to 76%, or 76% to 77%, or 77% to78%, or 78% to 79%, or 79% to 80%, or 80% to 81%, or 81% to 82%, or 82%to 83%, or 83% to 84%, or 84% to 85%, or 85% to 86%, or 86% to 87%, or87% to 88%, or 88% to 89%, or 89% to 90%, or 90% to 91%, or 91% to 92%,or 92% to 93%, or 93% to 94%, or 94% to 95%, or 95% to 96%, or 96% to97%, or 97% to 98%.

In an embodiment, the percent water content, by weight, in the tissuefillers described herein is about 1%, or about 2%, or about 3%, or about4%, or about 5%, or about 6%, or about 7%, or about 8%, or about 9%, orabout 10%, or about 11%, or about 12%, or about 13%, or about 14%, orabout 15%, or about 16%, or about 17%, or about 18%, or about 19%, orabout 20%, or about 21%, or about 22%, or about 23%, or about 24%, orabout 25%, or about 26%, or about 27%, or about 28%, or about 29%, orabout 30%, or about 31%, or about 32%, or about 33%, or about 34%, orabout 35%, or about 36%, or about 37%, or about 38%, or about 39%, orabout 40%, or about 41%, or about 42%, or about 43%, or about 44%, orabout 45%, or about 46%, or about 47%, or about 48%, or about 49%, orabout 50%, or about 51%, or about 52%, or about 53%, or about 54%, orabout 55%, or about 56%, or about 57%, or about 58%, or about 59%, orabout 60%, or about 61%, or about 62%, or about 63%, or about 64%, orabout 65%, or about 66%, or about 67%, or about 68%, or about 69%, orabout 70%, or about 71%, or about 72%, or about 73%, or about 74%, orabout 75%, or about 76%, or about 77%, or about 78%, or about 79%, orabout 80%, or about 81%, or about 82%, or about 83%, or about 84%, orabout 85%, or about 86%, or about 87%, or about 88%, or about 89%, orabout 90%, or about 91%, or about 92%, or about 93%, or about 94%, orabout 95%.

Mechanical Properties

The tissue fillers described herein, or components thereof, may beprovided in a number of physical states depending upon the selectedtherapy and mode of delivery. In some embodiments, the tissue fillers ofthe invention are fluids, for example liquids. In some embodiments, thetissue fillers of the invention are viscous fluids. In some embodiments,the tissue fillers of the invention are solids. In some embodiments, thetissue fillers of the invention are elastic solids.

A number of rheological properties may be evaluated when examining thetissue fillers described herein, as shown in Table 17:

TABLE 17 Rheology Terms Used to Describe Tissue Fillers ElasticityAbility of tissue filler to spring back to its original shape afterdeformation Elastic Modulus Measure of stored energy in viscoelasticmaterial represented by symbol G′ Viscosity Flow characteristics oftissue filler (gel thickness) Viscous Modulus Measure of dissipatedenergy in viscoelastic material represented by G″ Complex Modulus Totalresistance to deformation of tissue tiller determined by vector sum ofG′ and G″ (G*) Complex Viscosity Viscosity calculated from frequencysweep represented by n* Viscoelastic Describes tissue fillers whichpossess elastic and viscous properties Shear force External force whichis applied parallel to tissue filler by placing between two plates thatmist in opposite directions Shear thinning Decreasing tissue fillerviscosity with increasing rate of deformation.

In some embodiments, the tissue fillers of the invention areviscoelastic materials, which exhibit mechanical properties of bothelastic, and viscous materials. In some embodiments, the tissue fillersof the invention may be described as gels. Methods for assessing themechanical or rheological properties (e.g., viscoelastic properties) ofa material are known in the art, such as for example described in U.S.Patent Application Publication No. 2006/0105022 and Stocks, et al., J.Drugs. Dermatol. (2011) 10:974-980, the entirety of which areincorporated herein by reference. Viscoelasticity of a material can becharacterized by using dynamic mechanical analysis, for example byapplying an oscillatory stress to a sample and measuring the resultingstrain. Elastic materials typically exhibit in-phase stress and strain,i.e., application of stress results in immediate strain. In viscousmaterials, strain is de-phased from the application of stress by 90degrees. In viscoelastic materials, the phase difference between strainand stress is more than 0, but less than 90 degrees. In someembodiments, the viscoelasticity of SPF materials of the invention canbe characterized by means of the complex dynamic modulus G, whichincludes the storage modulus G′ (also referred to as the elasticmodulus), and the loss modulus G″ (also referred to as the viscousmodulus):

${{{{{G = {G^{\prime} + {iG}}}"}{{{{where}\mspace{14mu} i^{2}} = {- 1}},{G^{\prime} = {\frac{\sigma_{0}}{ɛ_{o}}\cos\mspace{11mu}\delta}},{{and}\mspace{14mu} G}}}"} = {\frac{\sigma_{0}}{ɛ_{o}}\sin\mspace{11mu}\delta}},$

σ₀ is the amplitude of stress, ε₀ is the amplitude of strain, and δ isthe phase shift.

The elastic modulus G′ and the loss modulus G″ are measured bysubjecting an SPF gel sample to an oscillatory stress in a rotational,or shear rheometer. The sample is placed between two plates, one fixedand one being able to rotate, or oscillate with a given frequency. Thevalues of the elastic modulus G′ and the loss modulus G″ are frequencydependent. Ranges of frequency used in measuring the elastic modulus G′and the loss modulus G″ are typically between, but not limited to, 0.1to 10 Hz. In some embodiments, the elastic modulus G′ and the lossmodulus G″ are measured at an oscillatory frequency of 1 Hz.

In some embodiments, rheological properties of the tissue fillersdescribed herein, e.g., G′ and G″, can be measured with an oscillatoryparallel plate rheometer. A plate of various diameters, for example 25mm can be used at a gap height between plates of various distances, forexample 1 mm. Measurements can be performed at various temperatures. Insome embodiments, measurements are performed at a constant temperatureof 25° C. In some embodiments, a measurement includes a frequency sweepbetween two frequency values, for example from 1 to 10 Hz, at a specificstrain value, for example at a constant strain of 2%. In someembodiments, measurements include a logarithmic increase of frequency,followed by a strain sweep which can be for example between 1 to 300% ata constant frequency, for example 5 Hz with a logarithmic increase instrain. In some embodiments, the storage modulus G′ and the loss modulusG″ can be obtained from a strain sweep at a specific percentage strainvalue, for example at 1% strain.

In some embodiments, the complex modulus (i.e., the sum of G′ and iG″)provides a comprehensive measure of total resistance to deformation of aparticular tissue filler described herein. Complex modulus may be testedusing a rheometer where a particular tissue filler (e.g., a gel) may besqueezed between two parallel circular plates and variable rotationalstrain is provided by rotating one plate at varying frequencies.

In some embodiments, the characteristics of a particular tissue fillermay be examined via that tissue filler's percent elasticity, wherepercent elasticity is equal to 100×G′/(G′+G″).

In some embodiments, the characteristics of a particular tissue fillermay be examined via that tissue filler's recovery coefficient:

${{Recovery}\mspace{14mu}{Coefficient}} = \frac{{Viscosity}\mspace{14mu}{value}\mspace{14mu}{obtained}\mspace{14mu}{during}\mspace{14mu}{increasing}\mspace{14mu}{sweep}\mspace{14mu}{frequency}}{{Viscosity}\mspace{14mu}{value}\mspace{14mu}{obtained}\mspace{14mu}{during}\mspace{14mu}{{de}{creasing}}\mspace{14mu}{sweep}\mspace{14mu}{frequency}}$

where: a recovery coefficient of about 1 means that the particulartissue filler (e.g., a gel) retained its structure despite appliedforces; a recovery coefficient of greater than 1 means that theparticular tissue filler (e.g., a gel) experienced structural breakdown;and a recovery coefficient of less than 1 gel experienced increasedstructural performance.

Without being limited to any one theory of the invention, increasing G′results in a relative increase in a material's ability to better resistalterations in shape and the material may be described as being firmer,harder, or more elastic than a material (e.g., gel tissue filler) with alower G′. Accordingly, increasing G′ may result in a correspondingincrease in a material's ability to provide structural support and/orvolumization.

Without being limited to any one theory of the invention, increasing G″results in a more viscous material (e.g., gel) as compared to a materialhaving a lower G″. Moreover, there is a greater energy loss asdissipated heat for materials with higher G″. In some embodiments, G′increases with an increasing degree of cross-linking. In someembodiments, G″ increases with an increasing degree of cross-linking. Insome embodiments, both G′ and G″ increase with an increasing degree ofcross-linking. In some embodiments, the tissue fillers of the inventionhave a G′ from about less than 50 Pa, to about more than 15000 Pa. Insome embodiments, the tissue fillers of the invention have a G′ fromabout 50 Pa to about 500.000 Pa. In some embodiments, the tissue fillersof the invention have a G′ from about 100 Pa to about 500.000 Pa. Insome embodiments, the tissue fillers of the invention have a G′ fromabout 75 Pa to about 150 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ from about 100 Pa to about 250 Pa. In someembodiments, the tissue fillers of the invention have a G′ from about150 Pa to about 275 Pa. In some embodiments, the tissue fillers of theinvention have a G′ from about 150 Pa to about 500 Pa. In someembodiments, the tissue fillers of the invention have a G′ from about250 Pa to about 750 Pa. In some embodiments, the tissue fillers of theinvention have a G′ from about 375 Pa to about 675 Pa. In someembodiments, the tissue fillers of the invention have a G′ from about425 Pa to about 850 Pa. In some embodiments, the tissue fillers of theinvention have a G′ from about 500 Pa to about 1000 Pa. In someembodiments, the tissue fillers of the invention have a G′ from about650 Pa to about 1050 Pa. In some embodiments, the tissue fillers of theinvention have a G′ from about 750 Pa to about 1250 Pa. In someembodiments, the tissue fillers of the invention have a G′ from about950 Pa to about 1500 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atleast 50 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 100 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at least 150 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at least 200 Pa, In someembodiments, the tissue fillers of the invention have a G′ of at least225 Pa, In some embodiments, the tissue fillers of the invention have aG′ of at least 250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 275 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 300 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least325 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 350 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 375 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 400 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least425 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 450 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 475 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 500 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atleast 525 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 550 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at least 575 Pa. In some embodiments, thetissue fillers of the invention have a G′ of about at least Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 625Pa. In some embodiments, the tissue fillers of the invention have a G′of at least 650 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 675 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 700 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least725 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 750 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 775 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 800 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least825 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 850 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 875 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 900 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least925 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 950 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 975 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 1000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atleast 1050 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 1100 Pa. In some embodiments, the tissue fillersof the invention have a G′ of at least 1150 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at least 1200 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least1250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 1300 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 1350 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 1400 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least1450 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 1500 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atmost 50 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at most 100 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at most 150 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at most 200 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most225 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 275 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 300 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most325 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 350 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 375 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 400 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most425 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 450 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 475 Pa, in some embodiments, the tissuefillers of the invention have a G′ of at most 500 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atmost 525 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at most 550 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at most 575 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at most Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 625Pa. In some embodiments, the tissue fillers of the invention have a G′of at most 650 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 675 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 700 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most725 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 750 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 775 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 800 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most825 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 850 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 875 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 900 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most925 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 950 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 975 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 1000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atmost 1050 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at most 1100 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at most 1150 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at most 1200 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most1250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 1300 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 1350 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 1400 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most1450 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 1500 Pa.

In some embodiments, the tissue fillers of the invention have a G′ ofabout 50 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of about 100 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of about 150 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 200 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 225 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 275 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 300 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 325 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 350 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 375Pa. In some embodiments, the tissue fillers of the invention have a G′of about 400 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 425 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 450 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 475 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 500Pa.

In some embodiments, the tissue fillers of the invention have a G′ ofabout 525 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of about 550 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of about 575 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 600 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 625 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 650Pa. In some embodiments, the tissue fillers of the invention have a G′of about 675 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 700 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 725 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 775Pa. In some embodiments, the tissue fillers of the invention have a G′of about 800 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 825 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 850 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 875 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 900Pa. In some embodiments, the tissue fillers of the invention have a G′of about 925 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 950 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 975 Pa. In some embodiments,the tissue fillers of the invention have a G′ of about 1000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ ofabout 1050 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of about 1100 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of about 1150 Pa. In some embodiments, thetissue fillers of the invention have a G′ of about 1200 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 1250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 1300 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 1350 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 1400 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 1450Pa. In some embodiments, the tissue fillers of the invention have a G′of about 1500 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atleast 2000 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 2250 Pa. In some embodiments, the tissue fillersof the invention have a G′ of at least 2500 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at least 2750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least3000 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 3250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 3500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 3750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least4000 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 4250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 4500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 4750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least5000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atleast 5250 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 5500 Pa. In some embodiments, the tissue fillersof the invention have a G′ of at least 5750 Pa. In some embodiments, thetissue fillers of the invention have a G′ of about at least 6000 Pa. Insome embodiments, the tissue fillers of the invention have a G′ of about6250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 6500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 6750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 7000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least7250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 7500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 7750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 8000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least8250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 8500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 8750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 9000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at least9250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at least 9500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at least 9750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at least 10000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atleast 10500 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 11000 Pa. In some embodiments, the tissue fillersof the invention have a G′ of at least 11500 Pa. In some embodiments,the tissue fillers of the invention have a G′ of at least 12000 Pa. Insome embodiments, the tissue fillers of the invention have a G′ of atleast 12500 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 13000 Pa. In some embodiments, the tissue fillersof the invention have a G′ of at least 13500 Pa. In some embodiments,the tissue fillers of the invention have a G′ of at least 14000 Pa. Insome embodiments, the tissue fillers of the invention have a G′ of atleast 14500 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at least 15000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atmost 2000 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at most 2250 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at most 2500 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at most 2750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most3000 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 3250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 3500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 3750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most4000 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 4250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 4500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 4750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most5000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atmost 5250 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at most 5500 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of at most 5750 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at most 6000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 6250Pa. In some embodiments, the tissue fillers of the invention have a G′of at most 6500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 6750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 7000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most7250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 7500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 7750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 8000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most8250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 8500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 8750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 9000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most9250 Pa. In some embodiments, the tissue fillers of the invention have aG′ of at most 9500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 9750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 10000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ of atmost 10500 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of at most 11000 Pa. In some embodiments, the tissue fillersof the invention have a G′ of at most 11500 Pa. In some embodiments, thetissue fillers of the invention have a G′ of at most 12000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most12500 Pa. In some embodiments, the tissue fillers of the invention havea G′ of at most 13000 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of at most 13500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of at most 14000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of at most14500 Pa. In some embodiments, the tissue fillers of the invention havea G′ of at most 15000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ ofabout 2000 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of about 2250 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of about 2500 Pa. In some embodiments, thetissue fillers of the invention have a G′ of about 2750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 3000Pa. In some embodiments, the tissue fillers of the invention have a G′of about 3250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 3500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 3750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 4000Pa. In some embodiments, the tissue fillers of the invention have a G′of about 4250 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 4500 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 4750 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 5000Pa.

In some embodiments, the tissue fillers of the invention have a G′ ofabout 5250 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of about 5500 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of about 5750 Pa. In some embodiments, thetissue fillers of the invention have a G′ of about 6000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 6250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 6500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 6750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 7000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 7250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 7500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 7750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 8000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 8250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 8500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 8750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 9000 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 9250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 9500 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 9750 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 10000 Pa.

In some embodiments, the tissue fillers of the invention have a G′ ofabout 1050 Pa. In some embodiments, the tissue fillers of the inventionhave a G′ of about 1100 Pa. In some embodiments, the tissue fillers ofthe invention have a G′ of about 1150 Pa. In some embodiments, thetissue fillers of the invention have a G′ of about 1200 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 1250Pa. In some embodiments, the tissue fillers of the invention have a G′of about 1300 Pa. In some embodiments, the tissue fillers of theinvention have a G′ of about 1350 Pa. In some embodiments, the tissuefillers of the invention have a G′ of about 1400 Pa. In someembodiments, the tissue fillers of the invention have a G′ of about 1450Pa. In some embodiments, the tissue fillers of the invention have a G′of about 1500 Pa.

In some embodiments, the tissue fillers of the invention have a G″ fromabout less than 5 Pa, to about more than 200 Pa. In some embodiments,the tissue fillers of the invention have a G″ from about 5 Pa to about200 Pa. In some embodiments, the tissue fillers of the invention have aG″ from about 5 Pa to about 25 Pa. In some embodiments, the tissuefillers of the invention have a G″ from about 15 Pa to about 35 Pa. Insome embodiments, the tissue fillers of the invention have a G″ fromabout 10 Pa to about 50 Pa. In some embodiments, the tissue fillers ofthe invention have a G″ from about 15 Pa to about 75 Pa. In someembodiments, the tissue fillers of the invention have a G″ from about Pato about 85 Pa. In some embodiments, the tissue fillers of the inventionhave a G″ from about 25 Pa to about 100 Pa. In some embodiments, thetissue fillers of the invention have a G″ from about 35 Pa to about 125Pa. In some embodiments, the tissue fillers of the invention have a G″from about 45 Pa to about 115 Pa. In some embodiments, the tissuefillers of the invention have a G″ from about 75 Pa to about 150 Pa. Insome embodiments, the tissue fillers of the invention have a G″ fromabout 100 Pa to about 175 Pa. In some embodiments, the tissue fillers ofthe invention have a G″ from about 115 Pa to about 200 Pa.

In some embodiments, the tissue fillers of the invention have a G″ of atleast 5 Pa. In some embodiments, the tissue fillers of the inventionhave a G″ of at least 10 Pa. In some embodiments, the tissue fillers ofthe invention have a G″ of at least 15 Pa. In some embodiments, thetissue fillers of the invention have a G″ of at least 20 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least25 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 30 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 35 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 40 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least45 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 50 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 55 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 60 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least65 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 70 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 75 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 80 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least85 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 90 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 95 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 100 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least105 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 110 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 115 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 120 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least125 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 130 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 135 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 140 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least145 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 150 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 155 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 160 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least165 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 170 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 175 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 180 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at least185 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at least 190 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at least 195 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at least 200 Pa.

In some embodiments, the tissue fillers of the invention have a G″ of atmost 5 Pa. In some embodiments, the tissue fillers of the invention havea G″ of at most 10 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 15 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 20 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most 25Pa. In some embodiments, the tissue fillers of the invention have a G″of at most 30 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 35 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 40 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most 45Pa. In some embodiments, the tissue fillers of the invention have a G″of at most 50 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 55 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 60 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most 65Pa. In some embodiments, the tissue fillers of the invention have a G″of at most 70 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 75 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 80 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most 85Pa. In some embodiments, the tissue fillers of the invention have a G″of at most 90 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 95 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 100 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most105 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at most 110 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 115 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 120 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most125 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at most 130 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 135 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 140 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most145 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at most 150 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 155 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 160 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most165 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at most 170 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 175 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 180 Pa. In someembodiments, the tissue fillers of the invention have a G″ of at most185 Pa. In some embodiments, the tissue fillers of the invention have aG″ of at most 190 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of at most 195 Pa. In some embodiments, the tissuefillers of the invention have a G″ of at most 200 Pa.

In some embodiments, the tissue fillers of the invention have a G″ ofabout 5 Pa. In some embodiments, the tissue fillers of the inventionhave a G″ of about 10 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of about 15 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 20 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 25 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 30Pa. In some embodiments, the tissue fillers of the invention have a G″of about 35 Pa. In some embodiments, the tissue fillers of the inventionhave a G″ of about 40 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of about 45 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 50 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 55 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 60Pa. In some embodiments, the tissue fillers of the invention have a G″of about 65 Pa. In some embodiments, the tissue fillers of the inventionhave a G″ of about 70 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of about 75 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 80 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 85 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 90Pa. In some embodiments, the tissue fillers of the invention have a G″of about 95 Pa. In some embodiments, the tissue fillers of the inventionhave a G″ of about 100 Pa. In some embodiments, the tissue fillers ofthe invention have a G″ of about 105 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 110 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 115 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 120Pa. In some embodiments, the tissue fillers of the invention have a G″of about 125 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of about 130 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 135 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 140 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 145Pa. In some embodiments, the tissue fillers of the invention have a G″of about 150 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of about 155 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 160 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 165 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 170Pa. In some embodiments, the tissue fillers of the invention have a G″of about 175 Pa. In some embodiments, the tissue fillers of theinvention have a G″ of about 180 Pa. In some embodiments, the tissuefillers of the invention have a G″ of about 185 Pa. In some embodiments,the tissue fillers of the invention have a G″ of about 190 Pa. In someembodiments, the tissue fillers of the invention have a G″ of about 195Pa. In some embodiments, the tissue fillers of the invention have a G″of about 200 Pa.

In some embodiments, a tissue filler disclosed herein exhibits dynamicviscosity. Viscosity is resistance of a fluid to shear or flow caused byeither shear stress or tensile stress. Viscosity describes a fluid'sinternal resistance to flow caused by intermolecular friction exertedwhen layers of fluids attempt to slide by one another and may be thoughtof as a measure of fluid friction. The less viscous the fluid, thegreater its ease of movement (fluidity).

Viscosity can be defined in two ways; dynamic viscosity (μ; η issometimes used) or kinematic viscosity (v). Dynamic viscosity, alsoknown as absolute or complex viscosity, is the tangential force per unitarea required to move one horizontal plane with respect to the other atunit velocity when maintained a unit distance apart by the fluid. The SIphysical unit of dynamic viscosity is the Pascal-second (Pas), which isidentical to Nm⁻²s. Dynamic viscosity can be expressed as τ=μdvx/dz,where τ=shearing stress, μ=dynamic viscosity, and dvx/dz is the velocitygradient over time. For example, if a fluid with a viscosity of one Pa·sis placed between two plates, and one plate is pushed sideways with ashear stress of one Pascal, it moves a distance equal to the thicknessof the layer between the plates in one second. Kinematic viscosity (v)is the ratio of dynamic viscosity to density, a quantity in which noforce is involved and is defined as follows: v=μ/ρ, where μ is thedynamic viscosity, and ρ is density (kg/m³). Kinematic viscosity isusually measured by a glass capillary viscometer as has an SI unit ofm²/s. The viscosity of a fluid is temperature dependent, and thusdynamic and kinematic viscosity are reported in reference totemperature.

In some embodiments, a tissue filler disclosed herein exhibits a dynamicviscosity of, for example, at least 10 Pa·s, at least 20 Pa·s, at least30 Pa·s, at least 40 Pa·s, at least 50 Pa·s, at least 60 Pa·s, at least70 Pa·s, at least 80 Pa·s, at least 90 Pa·s, at least 100 Pa·s, at least125 Pa·s, at least 150 Pa·s, at least 175 Pa·s, at least 200 Pas, atleast 225 Pa·s, at least 250 Pa·s, at least 275 Pa·s, at least 300 Pa·s,at least 400 Pa·s, at least 500 Pa·s, at least 600 Pa·s, at least 700Pa·s, at least 750 Pa·s, at least 800 Pa·s, at least 900 Pa·s, at least1,000 Pa·s, at least 1,100 Pa·s, or at least 1,200 Pa·s. In someembodiments, a tissue filler disclosed herein exhibits a dynamicviscosity of, for example, at most 10 Pa·s, at most Pa·s, at most 30Pa·s, at most 40 Pa·s, at most 50 Pa·s, at most 60 Pa·s, at most 70Pa·s, at most 80 Pa·s, at most 90 Pa·s, at most 100 Pa·s, at most 125Pa·s, at most 150 Pa·s, at most 175 Pa·s, at most 200 Pa·s, at most 225Pa·s, at most 250 Pa·s, at most 275 Pa·s, at most 300 Pa·s, at most 400Pa·s, at most 500 Pa·s, at most 600 Pa·s, at most 700 Pa·s, at most 750Pa·s, at most 800 Pa·s, at most 900 Pa·s, or at most 1000 Pa·s. In someembodiments, a tissue filler disclosed herein exhibits a dynamicviscosity of, for example, about 10 Pa·s to about 100 Pa·s, about 10Pa·s to about 150 Pa·s, about 10 Pa·s to about 250 Pa·s, about 50 Pa·sto about 100 Pa·s, about 50 Pa·s to about 150 Pa·s, about 50 Pa·s toabout 250 Pa·s, about 100 Pa·s to about 500 Pa·s, about 100 Pa·s toabout 750 Pa·s, about 100 Pa·s to about 1,000 Pa·s, about 100 Pa·s toabout 1,200 Pa·s, about 300 Pa·s to about 500 Pa·s, about 300 Pa·s toabout 750 Pa·s, about 300 Pa·s to about 1,000 Pa·s, or about 300 Pa·s toabout 1,200 Pa·s.

In an embodiment, the tissue fillers described herein may substantiallymaintain their G′ and/or G″ in vivo for at least 1 day, or at least 2days, or at least 3 days, or at least 4 days, or at least 5 days, or atleast 6 days, or at least 1 week, or at least 2 weeks, or at least 3weeks, or at least 1 month, or at least 2 months, or at least 3 months,or at least 4 months, or at least 5 months, or at least 6 months, or atleast 7 months, or at least 8 months, or at least 9 months, or at least10 months, or at least 11 months, or at least 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their G′ and/or G″ in vivo for at most 1 day, or at most 2days, or at most 3 days, or at most 4 days, or at most 5 days, or atmost 6 days, or at most 1 week, or at most 2 weeks, or at most 3 weeks,or at most 1 month, or at most 2 months, or at most 3 months, or at most4 months, or at most 5 months, or at most 6 months, or at most 7 months,or at most 8 months, or at most 9 months, or at most 10 months, or atmost 11 months, or at most 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their G′ and/or G″ in vivo for about 1 day, or about 2 days, orabout 3 days, or about 4 days, or about 5 days, or about 6 days, orabout 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, orabout 2 months, or about 3 months, or about 4 months, or about 5 months,or about 6 months, or about 7 months, or about 8 months, or about 9months, or about 10 months, or about 11 months, or about 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their elasticity in vivo for at least 1 day, or at least 2days, or at least 3 days, or at least 4 days, or at least 5 days, or atleast 6 days, or at least 1 week, or at least 2 weeks, or at least 3weeks, or at least 1 month, or at least 2 months, or at least 3 months,or at least 4 months, or at least 5 months, or at least 6 months, or atleast 7 months, or at least 8 months, or at least 9 months, or at least10 months, or at least 11 months, or at least 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their elasticity in vivo for at most 1 day, or at most 2 days,or at most 3 days, or at most 4 days, or at most 5 days, or at most 6days, or at most 1 week, or at most 2 weeks, or at most 3 weeks, or atmost 1 month, or at most 2 months, or at most 3 months, or at most 4months, or at most 5 months, or at most 6 months, or at most 7 months,or at most 8 months, or at most 9 months, or at most 10 months, or atmost 11 months, or at most 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their elasticity in vivo for about 1 day, or about 2 days, orabout 3 days, or about 4 days, or about 5 days, or about 6 days, orabout 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, orabout 2 months, or about 3 months, or about 4 months, or about 5 months,or about 6 months, or about 7 months, or about 8 months, or about 9months, or about 10 months, or about 11 months, or about 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their viscosity in vivo for at least 1 day, or at least 2 days,or at least 3 days, or at least 4 days, or at least 5 days, or at least6 days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, orat least 1 month, or at least 2 months, or at least 3 months, or atleast 4 months, or at least 5 months, or at least 6 months, or at least7 months, or at least 8 months, or at least 9 months, or at least 10months, or at least 11 months, or at least 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their viscosity in vivo for at most 1 day, or at most 2 days,or at most 3 days, or at most 4 days, or at most 5 days, or at most 6days, or at most 1 week, or at most 2 weeks, or at most 3 weeks, or atmost 1 month, or at most 2 months, or at most 3 months, or at most 4months, or at most 5 months, or at most 6 months, or at most 7 months,or at most 8 months, or at most 9 months, or at most 10 months, or atmost 11 months, or at most 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their viscosity in vivo for about 1 day, or about 2 days, orabout 3 days, or about 4 days, or about 5 days, or about 6 days, orabout 1 week, or about 2 weeks, or about 3 weeks, or about 1 month, orabout 2 months, or about 3 months, or about 4 months, or about 5 months,or about 6 months, or about 7 months, or about 8 months, or about 9months, or about 10 months, or about 11 months, or about 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their volume in vivo for at least 1 day, or at least 2 days, orat least 3 days, or at least 4 days, or at least 5 days, or at least 6days, or at least 1 week, or at least 2 weeks, or at least 3 weeks, orat least 1 month, or at least 2 months, or at least 3 months, or atleast 4 months, or at least 5 months, or at least 6 months, or at least7 months, or at least 8 months, or at least 9 months, or at least 10months, or at least 11 months, or at least 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their volume in vivo for at most 1 day, or at most 2 days, orat most 3 days, or at most 4 days, or at most 5 days, or at most 6 days,or at most 1 week, or at most 2 weeks, or at most 3 weeks, or at most 1month, or at most 2 months, or at most 3 months, or at most 4 months, orat most 5 months, or at most 6 months, or at most 7 months, or at most 8months, or at most 9 months, or at most 10 months, or at most 11 months,or at most 1 year.

In an embodiment, the tissue fillers described herein may substantiallymaintain their volume in vivo for about 1 day, or about 2 days, or about3 days, or about 4 days, or about 5 days, or about 6 days, or about 1week, or about 2 weeks, or about 3 weeks, or about 1 month, or about 2months, or about 3 months, or about 4 months, or about 5 months, orabout 6 months, or about 7 months, or about 8 months, or about 9 months,or about 10 months, or about 11 months, or about 1 year.

Methods of Manufacture

The tissue fillers provided herein may be prepared by combining an SPFbased component with an HA based component with or without anyadditional agents. In certain embodiments, one or both of the SPF and HAmay be cross-linked prior to combination. In some embodiments, the SPFand HA may be combined and then cross-linked with a cross-linking agentas described herein. In some embodiments, the SPF may be cross-linkedwith a cross linking agent and then added to a HA, which may or may notbe cross linked, and then the combination thereof may be subjected toadditional cross linking. In some embodiments, the HA may becross-linked with a cross linking agent and then added to a SPF, whichmay or may not be cross linked, and then the combination thereof may besubjected to additional cross linking.

In some embodiments, the tissue fillers described herein may be preparedby combining an SPF based component, and HA based component, and anadditional agent, as described hereinabove. In such embodiments, one orboth of the SPF and HA may be cross-linked prior to combination. In someembodiments, the SPF and HA may be combined with the additional agentand then cross-linked with a cross-linking agent as described herein. Insome embodiments, the additional agent may be added after combining theSPF and HA.

In some embodiments, the tissue filler described herein may include SPFand HA in a weight ratio (SPF:HA) of 0.1:1 to 0.1:10, or 0.1:1 to0.1:100, or 0.1:1000; 1:1 to 1:10, or 1:1 to 1:100, or 1:1 to 1:1000.

In some embodiments, the tissue filler described herein may include SPFand HA in a weight ratio (HA:SPF) of 0.1:1 to 0.1:10, or 0.1:1 to0.1:100, or 0.1:1000; 1:1 to 1:10, or 1:1 to 1:100, or 1:1 to 1:1000.

In some embodiments, a resulting HA/SPF combination (whethercross-linked or non-cross-linked) may be homogenized such as throughmechanical blending of initially cross-linked HA and/or SPF.

In some embodiments, a solution of SPF may be provided and cross-linkedwith a cross linking agent to yield a cross-linked SPF, to which HA maybe added in either its cross-linked form, non-cross-linked form, or amixture thereof. The resulting mixture may then be homogenized and anyadditional agents (e.g., lidocaine may be added).

In some embodiments, a solution of SPF may be provided and cross-linkedwith a cross linking agent in the presence of HA to yield a cross-linkedSPF-HA composition, to which HA may, or may not, be added in itsnon-cross-linked form. The resulting mixture may then be homogenized andany additional agents (e.g., lidocaine may be added).

In some embodiments the specific SPF formulations provided herein may becombined with HA, or may utilize the cross-linking procedures, using thepreparations set forth in U.S. Pat. Nos. 8,288,347 or 8,450,475, or U.S.Patent Application Publication Nos. 2006/0105022, 2016/0376382, or2017/0315828, the entirety of which are incorporated herein byreference.

In some embodiments, the methods described herein may include asterilization step where the tissue filler or a portion thereof isexposed, for example, to temperatures of 120° C. to about 130° C. andpressures of about 12 to about 20 pounds per square inch for a time ofabout 1 to about 15 minutes.

In some embodiments, the methods described herein may include ade-gassing step wherein the SPF, HA, or SPF/HA solutions describedherein that are used in preparing the resulting tissue fillers arede-gassed.

In some embodiments, the tissue fillers described herein may be preparedaccording to the general methods described in Examples 5 to 20. In themethods described therein, silk may be prepared in an aqueous solution,an aqueous/alcohol solution, wherein the alcohol may be ethanol ormethanol, for example. In the methods described therein, any of thecrosslinking agents described herein may be used as applicable to crosslink SPF to SPF, SPF to HA, or HA to HA, as would be understood by aperson having ordinary skill in art.

Methods of Treatment

In an embodiment, the tissue fillers described herein may be provided inmethods of treating one or more conditions in a patient in need thereof.In some embodiments, a therapeutically effective amount of a tissuefiller may be delivered into a tissue of a patient in need thereof totreat a condition or other tissue deficiency.

As used herein, the term “treating,”, “treat”, or “treatment” refers toreducing or eliminating in a patient a cosmetic or clinical symptom of acondition, such as a soft tissue condition, or delaying or preventing inan individual the onset of a cosmetic or clinical symptom of acondition.

In some embodiments, the condition treated by the tissue fillersdescribed herein may include a soft tissue condition. Soft tissueconditions include, without limitation, augmentations, reconstructions,diseases, disorders, defects, or imperfections of a body part, region orarea. In one aspect, a soft tissue condition treated by the disclosedtissue fillers include, without limitation, a facial augmentation, afacial reconstruction, a facial disease, a facial disorder, a facialdefect, or a facial imperfection. In some embodiments, a soft tissuecondition treated by the tissue fillers described herein include,without limitation, skin dehydration, a lack of skin elasticity, skinroughness, a lack of skin tautness, a skin stretch line or mark, skinpaleness, a dermal divot, a sunken check, a sunken temple, a thin lip, aurethra defect, a skin defect, a breast defect, a retro-orbital defect,a facial fold, or a wrinkle. In some embodiments, a soft tissuecondition treated by the tissue fillers described herein include,without limitation, breast imperfection, defect, disease and/ordisorder, such as, e.g., a breast augmentation, a breast reconstruction,mastopexy, micromastia, thoracic hypoplasia, Poland's syndrome, defectsdue to implant complications like capsular contraction and/or rupture; afacial imperfection, defect, disease or disorder, such as, e.g., afacial augmentation, a facial reconstruction. Parry-Romberg syndrome,lupus erythematosus profundus, dermal divots, sunken cheeks, sunkentemples, thin lips, nasal imperfections or defects, retro-orbitalimperfections or defects, a facial fold, line and/or wrinkle like aglabellar line, a nasolabial line, a perioral line, and/or a marionetteline, and/or other contour deformities or imperfections of the face; aneck imperfection, defect, disease or disorder; a skin imperfection,defect, disease and/or disorder; other soft tissue imperfections,defects, diseases and/or disorders, such as, e.g., an augmentation or areconstruction of the upper arm, lower arm, hand, shoulder, back, torsoincluding abdomen, buttocks, upper leg, lower leg including calves, footincluding plantar fat pad, eye, genitals, or other body part, region orarea, or a disease or disorder affecting these body parts, regions orareas; urinary incontinence, fecal incontinence, other forms ofincontinence; and gastroesophageal reflux disease (GERD).

In some embodiments, the tissue fillers described herein may bedelivered to soft tissues including, without limitation skin, dermaltissues, subdermal tissues, cutaneous tissues, subcutaneous tissues,intradural tissue, muscles, tendons, ligaments, fibrous tissues, fat,blood vessels and arteries, nerves, and synovial (intradermal) tissues.

In some embodiments, the tissue fillers described herein can be placeddirectly in a wound to aid in healing by providing an artificialbiodegradable matrix along with cell attachment, migration, andproliferation signals. In some embodiments, the tissue fillers describedherein can be coated on a biodegradable mesh or other implantedmaterial, or it can itself be formed into sheets or other structures, orcan be maintained in a hydrated form.

In some embodiments, the amount of a composition used with any of themethods as disclosed herein will be determined based on the alterationand/or improvement desired, the reduction and/or elimination of acondition symptom desired, the clinical and/or cosmetic effect desiredby the individual and/or physician, and the body part or region beingtreated. The effectiveness of composition administration may bemanifested by one or more of the following clinical and/or cosmeticmeasures: altered and/or improved soft tissue shape, altered and/orimproved soft tissue size, altered and/or improved soft tissue contour,altered and/or improved tissue function, tissue ingrowth support and/ornew collagen deposition, sustained engraftment of the tissue filler,improved patient satisfaction and/or quality of life, and decreased useof implantable foreign material. For example, for breast augmentationprocedures, effectiveness of the compositions and methods may bemanifested by one or more of the following clinical and/or cosmeticmeasures: increased breast size, altered breast shape, altered breastcontour, sustained engraftment, reduction in the risk of capsularcontraction, decreased rate of liponecrotic cyst formation, improvedpatient satisfaction and/or quality of life, and decreased use of breastimplant.

In some embodiments, effectiveness of the tissue fillers and methods intreating a facial soft tissue may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased size, shape,and/or contour of facial feature like increased size, shape, and/orcontour of lip, cheek, temple, or eye region; altered size, shape,and/or contour of facial feature like altered size, shape, and/orcontour of lip, cheek, temple, or eye region shape; reduction orelimination of a wrinkle, fold or line in the skin; resistance to awrinkle, fold or line in the skin; rehydration of the skin; increasedelasticity to the skin; reduction or elimination of skin roughness;increased and/or improved skin tautness; reduction or elimination ofstretch lines or marks; increased and/or improved skin tone, shine,brightness and/or radiance; increased and/or improved skin color,reduction or elimination of skin paleness; sustained engraftment ofcomposition; decreased side effects; improved patient satisfactionand/or quality of life.

In some embodiments, the invention provides for tissue fillers andmethods of treatment involving a dermal region. As used herein, the term“dermal region” refers to the region of skin comprising theepidermal-dermal junction and the dermis including the superficialdermis (papillary region) and the deep dermis (reticular region). Theskin is composed of three primary layers: the epidermis, which provideswaterproofing and serves as a barrier to infection; the dermis, whichserves as a location for the appendages of skin; and the hypodermis(subcutaneous adipose layer). The epidermis contains no blood vessels,and is nourished by diffusion from the dermis. The main type of cellswhich make up the epidermis are keratinocytes, melanocytes, Langerhanscells, and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the stratum basal of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Stretch marks frompregnancy are for example located in the dermis.

The hypodermis lies below the dermis. Its purpose is to attach thedermal region of the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

In some embodiments, a tissue filler disclosed herein is administered toa skin region of an individual by injection into a dermal region or ahypodermal region. In some embodiments, a tissue filler disclosed hereinis administered to a dermal region of an individual by injection into,e.g., an epidermal-dermal junction region, a papillary region, areticular region, or any combination thereof.

In some embodiments, the invention provides methods of treating a softtissue condition of an individual, including administering one or moretissue fillers disclosed herein to a site of the soft tissue conditionof the individual, wherein the administration of the compositionimproves the soft tissue condition, thereby treating the soft tissuecondition. In some embodiments, a soft tissue condition is a breasttissue condition, a facial tissue condition, a neck condition, a skincondition, an upper arm condition, a lower arm condition, a handcondition, a shoulder condition, a back condition, a torso includingabdominal condition, a buttock condition, an upper leg condition, alower leg condition including calf condition, a foot condition includingplantar fat pad condition, an eye condition, a genital condition, or acondition effecting another body part, region or area.

In some embodiments, the invention provides methods of treating a skincondition including administering to an individual suffering from a skincondition one or more tissue fillers disclosed herein, wherein theadministration of the tissue filler improves the skin condition, therebytreating the skin condition. In some embodiments, a skin conditionincludes skin dehydration, and the method of treatment includesadministering to an individual suffering from skin dehydration one ormore tissue fillers disclosed herein, wherein the administration of thetissue filler rehydrates the skin, thereby treating skin dehydration. Inanother aspect of these embodiments, a method of treating a lack of skinelasticity includes administering to an individual suffering from a lackof skin elasticity a tissue filler disclosed herein, wherein theadministration of the tissue filler increases the elasticity of theskin, thereby treating a lack of skin elasticity. In yet another aspectof these embodiments, a method of treating skin roughness includesadministering to an individual suffering from skin roughness acomposition disclosed herein, wherein the administration of thecomposition decreases skin roughness, thereby treating skin roughness.In some embodiments, a method of treating a lack of skin tautnessincludes administering to an individual suffering from a lack of skintautness a tissue filler disclosed herein, wherein the administration ofthe tissue filler makes the skin tauter, thereby treating a lack of skintautness.

In some embodiments, the invention provides methods of treating a skinstretch line or mark, including administering to an individual sufferingfrom a skin stretch line or mark one or more tissue fillers disclosedherein, wherein the administration of the one or more tissue fillersreduces or eliminates the skin stretch line or mark, thereby treating askin stretch line or mark. In some embodiments, a method of treatingskin paleness includes administering to an individual suffering fromskin paleness a tissue filler disclosed herein, wherein theadministration of the tissue filler increases skin tone or radiance,thereby treating skin paleness. In some embodiments, a method oftreating skin wrinkles includes administering to an individual sufferingfrom skin wrinkles a tissue filler disclosed herein, wherein theadministration of the tissue filler reduces or eliminates skin wrinkles,thereby treating skin wrinkles. In yet another aspect of theseembodiments, a method of treating skin wrinkles includes administeringto an individual a tissue filler disclosed herein, wherein theadministration of the tissue filler makes the skin resistant to skinwrinkles, thereby treating skin wrinkles.

In some embodiments, the invention provides administration of acomposition disclosed herein wherein such administration promotes newcollagen deposition or formation. The tissue fillers described hereinmay support tissue ingrowth and new deposition or formation of collagen.

Without being limited to any one theory of the invention, the molecularweight of SPFs used in the preparation tissue fillers described hereinmay be adjusted to provide a mild inflammatory response at a selectedtissue in order trigger the deposition or formation of collagen throughthe resulting tissue proliferation and maturation responses that followthe initial inflammatory response. Indeed, higher molecular weight SPFsmay result in an increased inflammatory response while lower molecularweight SPFs may result in little or no inflammatory response.

Without being limited to any one theory of the invention, the tissuefillers described herein provide the unexpected attribute that aresulting inflammatory response, and thereby collagen formation throughthe proliferation and maturation tissue response, may be tuned becausethe SPF solutions used herein have narrow rather than broadpolydispersities. In an embodiment, administration of a tissue fillerdisclosed herein increases new collagen deposition.

In some embodiments, administration of a tissue disclosed hereinincreases new collagen deposition or formation by about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 200%, about 21%, about 22%,about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%,about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%,about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%,about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%,about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%,about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about100%, relative to the same or similar tissue filler comprising HA, butlacking SPF.

In some embodiments, administration of a tissue filler disclosed hereinincreases new collagen deposition or formation by at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, atleast 13%, at least 14%, at least 15%, at least 16%, at least 17%, atleast 18%, at least 19%, at least 20%, at least 21%, at least 22%, atleast 23%, at least 24%, at least 25%, at least 26%, at least 27%, atleast 28%, at least 29%, at least 30%, at least 31%, at least 32%, atleast 33%, at least 34%, at least 35%, at least 36%, at least 37%, atleast 38%, at least 39%, at least 40%, at least 41%, at least 42%, atleast 43%, at least 44%, at least 45%, at least 46%, at least 47%, atleast 48%, at least 49%, at least 50%, at least 51%, at least 52%, atleast 53%, at least 54%, at least 55%, at least 56%, at least 57%, atleast 58%, at least 59%, at least 60%, at least 61%, at least 62%, atleast 63%, at least 64%, at least 65%, at least 66%, at least 67%, atleast 68%, at least 69%, at least 70%, at least 71%, at least 72%, atleast 73%, at least 74%, at least 75%, at least 76%, at least 77%, atleast 78%, at least 79%, at least 80%, at least 81%, at least 82%, atleast 83%, at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, at least 100%, at least 125%, at least 150%, atleast 175%, at least 200%, at least 225%, at least 250%, at least 275%,or at least 300%, relative to the same or similar tissue fillercomprising HA, but lacking SPF.

In some embodiments, administration of a tissue filler disclosed hereinincreases new collagen deposition or formation by at most 1%, at most2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, at most8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%, atmost 14%, at most 15%, at most 16%, at most 17%, at most 18%, at most19%, at most 20%, at most 21%, at most 22%, at most 23%, at most 24%, atmost 25%, at most 26%, at most 27%, at most 28%, at most 29%, at most30%, at most 31%, at most 32%, at most 33%, at most 34%, at most 35%, atmost 36%, at most 37%, at most 38%, at most 39%, at most 40%, at most41%, at most 42%, at most 43%, at most 44%, at most 45%, at most 46%, atmost 47%, at most 48%, at most 49%, at most 50%, at most 51%, at most52%, at most 53%, at most 54%, at most 55%, at most 56%, at most 57%, atmost 58%, at most 59%, at most 60%, at most 61%, at most 62%, at most63%, at most 64%, at most 65%, at most 66%, at most 67%, at most 68%, atmost 69%, at most 70%, at most 71%, at most 72%, at most 73%, at most74%, at most 75%, at most 76%, at most 770%, at most 78%, at most 79%,at most 80%, at most 81%, at most 82%, at most 83%, at most 84%, at most85%, at most 86%, at most 87%, at most 88%, at most 89%, at most 90%, atmost 91%, at most 92%, at most 93%, at most 94%, at most 95%, at most96%, at most 97%, at most 98%, at most 99%, at most 100%, at most 125%,at most 150%, at most 175%, at most 200%, at most 225%, at most 250%, atmost 275%, or at most 300%, relative to the same or similar tissuefiller comprising HA, but lacking SPF.

In some embodiments, administration of a tissue filler disclosed hereinincreases new collagen deposition or formation by about 1% to about 10%,about 10% to about 50%, about 10% to about 100%, about 50% to about150%, about 100% to about 200%, about 150% to about 250%, about 200% toabout 300%, about 350% to about 450%, about 400% to about 500%, about550% to about 650%, about 600% to about 700%, relative to the same orsimilar tissue filler comprising HA, but lacking SPF.

In some embodiments, the amount of a tissue filler used with any of themethods disclosed herein will typically be a therapeutically effectiveamount. As used herein, the term “therapeutically effective amount” issynonymous with “effective amount”, “therapeutically effective dose”,and/or “effective dose,” and refers to the amount of tissue filler thatwill elicit the expected biological, cosmetic, or clinical response in apatient in need thereof. As a non-limiting example, an effective amountis an amount sufficient to achieve one or more of the clinical and/orcosmetic measures disclosed herein. The appropriate effective amount tobe administered for a particular application of the disclosed methodscan be determined by those skilled in the art, using the guidanceprovided herein. For example, an effective amount can be extrapolatedfrom any and all in vitro and in vivo assays as described herein. Oneskilled in the art will recognize that the condition of the individualcan be monitored throughout the course of therapy and that the effectiveamount of a composition disclosed herein that is administered can beadjusted accordingly.

In some embodiments, the amount of a tissue filler administered is atleast 0.001 g, or at least 0.002 g, or at least 0.003 g, or at least0.004 g, or at least 0.005 g, or at least 0.006 g, or at least 0.007 g,or at least 0.008 g, or at least 0.009 g, or at least 0.01 g, or atleast 0.02 g, or at least 0.03 g, or at least 0.04 g, or at least 0.05g, or at least 0.06 g, or at least 0.07 g, or at least 0.08 g, or atleast 0.09 g, or at least 0.1 g, or at least 0.2 g, or at least 0.3 g,or at least 0.4 g, or at least 0.5 g, or at least 0.6 g, or at least 0.7g, or at least 0.8 g, or at least 0.9 g, or at least 1 g, or at least 2g, or at least 3 g, or at least 4 g, or at least 5 g, or at least 6 g,or at least 7 g, or at least 8 g, or at least 9 g, or at least 10 g, orat least 11 g, or at least 12 g, or at least 13 g, or at least 14 g, orat least 15 g, or at least 20 g, or at least 25 g, or at least 30 g, orat least 35 g, or at least 40 g, or at least 45 g, or at least 50 g, orat least 55 g, or at least 60 g, or at least 65 g, or at least 70 g, orat least 75 g, or at least 80 g, or at least 85 g, or at least 90 g, orat least 95 g, or at least 100 g.

In some embodiments, the amount of a tissue filler administered is atmost 0.001 g, or at most 0.002 g, or at most 0.003 g, or at most 0.004g, or at most 0.005 g, or at most 0.006 g, or at most 0.007 g, or atmost 0.008 g, or at most 0.009 g, or at most 0.01 g, or at most 0.02 g,or at most 0.03 g, or at most 0.04 g, or at most 0.05 g, or at most 0.06g, or at most 0.07 g, or at most 0.08 g, or at most 0.09 g, or at most0.1 g, or at most 0.2 g, or at most 0.3 g, or at most 0.4 g, or at most0.5 g, or at most 0.6 g, or at most 0.7 g, or at most 0.8 g, or at most0.9 g, or at most 1 g, or at most 2 g, or at most 3 g, or at most 4 g,or at most 5 g, or at most 6 g, or at most 7 g, or at most 8 g, or atmost 9 g, or at most 10 g, or at most 11 g, or at most 12 g, or at most13 g, or at most 14 g, or at most 15 g, or at most 20 g, or at most 25g, or at most 30 g, or at most 35 g, or at most 40 g, or at most 45 g,or at most 50 g, or at most 55 g, or at most 60 g, or at most 65 g, orat most 70 g, or at most 75 g, or at most 80 g, or at most 85 g, or atmost 90 g, or at most 95 g, or at most 100 g.

In some embodiments, the amount of a tissue filler administered is about0.001 g, or about 0.002 g, or about 0.003 g, or about 0.004 g, or about0.005 g, or about 0.006 g, or about 0.007 g, or about 0.008 g, or about0.009 g, or about 0.01 g, or about 0.02 g, or about 0.03 g, or about0.04 g, or about 0.05 g, or about 0.06 g, or about 0.07 g, or about 0.08g, or about 0.09 g, or about 0.1 g, or about 0.2 g, or about 0.3 g, orabout 0.4 g, or about 0.5 g, or about 0.6 g, or about 0.7 g, or about0.8 g, or about 0.9 g, or about 1 g, or about 2 g, or about 3 g, orabout 4 g, or about 5 g, or about 6 g, or about 7 g, or about 8 g, orabout 9 g, or about 10 g, or about 11 g, or about 12 g, or about 13 g,or about 14 g, or about 15 g, or about 20 g, or about 25 g, or about 30g, or about 35 g, or about 40 g, or about 45 g, or about 50 g, or about55 g, or about 60 g, or about 65 g, or about 70 g, or about 75 g, orabout 80 g, or about 85 g, or about 90 g, or about 95 g, or about 100 g.

In some embodiments, the amount of a tissue filler administered is 0.001g to 0.01 g, or 0.01 g to 0.1 g, or 0.1 g to 1 g, or 1 g to 10 g, or 10g to 20 g, or 20 g to 30 g, or 30 g to 40 g, or 40 g to 50 g, or 50 g to60 g, or 60 g to 70 g, or 70 g to 80 g, or 80 g to 90 g, or 90 g to 100g.

In some embodiments, the volume of a tissue filler administered is atleast 0.01 mL, or at least 0.02 mL, or at least 0.03 niL, or at least0.04 mL, or at least 0.05 mL, or at least 0.06 mL, or at least 0.07 mL,or at least 0.08 mL, or at least 0.09 mL, or at least 0.10 mL, or atleast 0.15 mL, or at least 0.20 mL, or at least 0.25 mL, or at least0.30 mL, or at least 0.35 mL, or at least 0.40 mL, or at least 0.45 mL,or at least 0.50 mL, or at least 0.55 mL, or at least 0.60 mL, or atleast 0.65 niL, or at least 0.70 mL, or at least 0.75 mL, or at least0.80 mL, or at least 0.85 mL, or at least 0.90 mL, or at least 0.95 mL,or at least 1 mL, or at least 2 niL, or at least 3 mL, or at least 4 mL,or at least 5 mL, or at least 6 mL, or at least 7 mL, or at least, 8 mL,or at least 9 mL, or at least 10 mL, or at least 15 mL, or at least 20mL, or at least 25 mL, or at least 30 mL, or at least 35 mL, or at least40 mL, or at least 45 mL, or at least 50 mL, or at least 55 niL, or atleast 60 mL, or at least 65 mL, or at least 70 mL, or at least 75 mL, orat least 80 mL, or at least 85 mL, or at least 90 mL, or at least 95 mL,or at least 100 mL, or at least 110 mL, or at least 120 mL, or at least130 mL, or at least 140 mL, or at least 150 mL, or at least 160 mL, orat least 170 mL, or at least 180 mL, or at least 190 mL, or at least 200mL, or at least 210 mL, or at least 220 mL, or at least 230 mL, or atleast 240 mL, or at least 250 mL, or at least 260 mL, or at least 270mL, or at least 280 mL, or at least 290 mL, or at least 300 mL, or atleast 325, 350 mL, or at least 375 mL, or at least 400 mL, or at least425 mL, or at least 450 mL, or at least 475 mL, or at least 500 mL, orat least 525 mL, or at least 550 mL, or at least 575 mL, or at least 600mL, or at least 625 mL, or at least 650 mL, or at least 675 niL, or atleast 700 mL, or at least 725 mL, or at least 750 mL, or at least 775mL, or at least 800 mL, or at least 825 mL, or at least 850 mL, or atleast 875 mL, or at least 900 mL, or at least 925 mL, or at least 950mL, or at least 975 mL, or at least 1000 mL.

In some embodiments, the volume of a tissue filler administered is atmost 0.01 mL, or at most 0.02 mL, or at most 0.03 mL, or at most 0.04mL, or at most 0.05 mL, or at most 0.06 mL, or at most 0.07 mL, or atmost 0.08 mL, or at most 0.09 mL, or at most 0.10 mL, or at most 0.15mL, or at most 0.20 mL, or at most 0.25 mL, or at most 0.30 mL, or atmost 0.35 mL, or at most 0.40 mL, or at most 0.45 mL, or at most 0.50mL, or at most 0.55 mL, or at most 0.60 mL, or at most 0.65 mL, or atmost 0.70 mL, or at most 0.75 mL, or at most 0.80 mL, or at most 0.85mL, or at most 0.90 mL, or at most 0.95 mL, or at most 1 mL, or at most2 mL, or at most 3 mL, or at most 4 mL, or at most 5 mL, or at most 6mL, or at most 7 mL, or at most, 8 mL, or at most 9 mL, or at most 10mL, or at most 15 mL, or at most 20 mL, or at most 25 mL, or at most 30mL, or at most 35 mL, or at most 40 mL, or at most 45 mL, or at most 50mL, or at most 55 mL, or at most 60 mL, or at most 65 mL, or at most 70mL, or at most 75 mL, or at most 80 mL, or at most 85 mL, or at most 90mL, or at most 95 mL, or at most 100 mL, or at most 110 mL, or at most120 mL, or at most 130 mL, or at most 140 mL, or at most 150 mL, or atmost 160 mL, or at most 170 mL, or at most 180 mL, or at most 190 mL, orat most 200 mL, or at most 210 mL, or at most 220 mL, or at most 230 mL,or at most 240 mL, or at most 250 mL, or at most 260 mL, or at most 270mL, or at most 280 mL, or at most 290 mL, or at most 300 mL, or at most325, 350 mL, or at most 375 mL, or at most 400 mL, or at most 425 mL, orat most 450 mL, or at most 475 mL, or at most 500 mL, or at most 525 mL,or at most 550 mL, or at most 575 mL, or at most 600 mL, or at most 625mL, or at most 650 mL, or at most 675 mL, or at most 700 mL, or at most725 mL, or at most 750 mL, or at most 775 mL, or at most 800 mL, or atmost 825 mL, or at most 850 mL, or at most 875 mL, or at most 900 mL, orat most 925 mL, or at most 950 mL, or at most 975 mL, or at most 1000mL.

In some embodiments, the volume of a tissue filler administered is about0.01 mL, or about 0.02 mL, or about 0.03 mL, or about 0.04 mL, or about0.05 mL, or about 0.06 mL, or about 0.07 mL, or about 0.08 mL, or about0.09 mL, or about 0.10 mL, or about 0.15 mL, or about 0.20 mL, or about0.25 mL, or about 0.30 mL, or about 0.35 mL, or about 0.40 mL, or about0.45 mL, or about 0.50 mL, or about 0.55 mL, or about 0.60 mL, or about0.65 mL, or about 0.70 mL, or about 0.75 mL, or about 0.80 mL, or about0.85 mL, or about 0.90 mL, or about 0.95 mL, or about 1 mL, or about 2mL, or about 3 mL, or about 4 mL, or about 5 mL, or about 6 mL, or about7 mL, or about, 8 mL, or about 9 mL, or about 10 mL, or about 11 mL, orabout 12 mL, or about 13 mL, or about 14 mL, or about 15 niL, or about16 mL, or about 17 mL, or about 18 niL, or about 19 mL, or about 20 mL,or about 21 mL, or about 22 mL, or about 23 mL, or about 24 mL, or about25 mL, or about 26 mL, or about 27 mL, or about 28 niL, or about 30 mL,or about 35 mL, or about 36 mL, or about 37 mL, or about 38 mL, or about39 mL, or about 40 mL, or about 41 mL, or about 42 mL, or about 43 mL,or about 44 mL, or about 45 mL, or about 46 mL, or about 47 mL, or about48 mL, or about 49 mL, or about 50 mL, or about 51 mL, or about 52 mL,or about 53 mL, or about 54 mL, or about 55 niL, or about 56 mL, orabout 57 mL, or about 58 mL, or about 59 mL, or about 60 mL, or about 61niL, or about 62 mL, or about 63 mL, or about 64 mL, or about 65 mL, orabout 66 mL, or about 67 mL, or about 68 mL, or about 69 mL, or about 70mL, or about 71 mL, or about 72 mL, or about 73 mL, or about 74 mL, orabout 75 mL, or about 76 niL, or about 77 mL, or about 78 mL, or about79 mL, or about 80 mL, or about 81 mL, or about 82 mL, or about 83 mL,or about 84 mL, or about 85 mL, or about 86 mL, or about 87 mL, or about88 mL, or about 89 mL, or about 90 mL, or about 91 niL, or about 92 mL,or about 93 mL, or about 94 mL, or about 95 mL, or about 96 mL, or about97 mL, or about 98 mL, or about 99 mL, or about 100 mL, or about 110niL, or about 120 mL, or about 130 mL, or about 140 mL, or about 150 mL,or about 160 mL, or about 170 mL, or about 180 mL, or about 190 mL, orabout 200 mL, or about 210 mL, or about 220 mL, or about 230 mL, orabout 240 mL, or about 250 mL, or about 260 mL, or about 270 mL, orabout 280 mL, or about 290 mL, or about 300 mL, or about 310 mL, orabout 320 mL, or about 330 mL, or about 340 mL, or about 350 mL, orabout 360 niL, or about 370 mL, or about 380 mL, or about 390 mL, orabout 400 mL, or about 410 mL, or about 420 mL, or about 430 mL, orabout 440 mL, or about 450 mL, or about 460 mL, or about 470 mL, orabout 480 mL, or about 490 mL, or about 500 mL, or about 510 mL, orabout 520 mL, or about 530 mL, or about 540 mL, or about 550 mL, orabout 560 niL, or about 570 mL, or about 580 mL, or about 590 mL, orabout 600 mL, or about 610 mL, or about 620 mL, or about 630 mL, orabout 640 mL, or about 650 mL, or about 660 mL, or about 670 mL, orabout 680 mL, or about 690 mL, or about 700 mL, or about 710 mL, orabout 720 mL, or about 730 mL, or about 740 mL, or about 750 mL, orabout 760 mL, or about 770 mL, or about 780 mL, or about 790 mL, orabout 800 mL, or about 810 niL, or about 820 mL, or about 830 mL, orabout 840 mL, or about 850 mL, or about 860 mL, or about 870 mL, orabout 880 mL, or about 890 mL, or about 900 mL, or about 910 mL, orabout 920 mL, or about 930 mL, or about 940 mL, or about 950 mL, orabout 960 mL, or about 970 mL, or about 980 mL, or about 990 mL, orabout 1000 mL.

In some embodiments, the volume of a tissue filler administered is 0.01mL to 0.10 mL, or 0.10 mL to 1 mL, or 1 mL to 10 mL, or 10 mL to 100 mL,or 50 mL to 100 mL, or 100 mL to 150 mL, or 150 mL to 200 mL, or 200 mLto 250 mL, or 250 mL to 300 mL, or 300 mL to 350 mL, or 350 mL to 400mL, or 400 mL to 450 mL, or 450 mL to 500 mL, or 500 mL to 550 mL, or550 mL to 600 mL, or 600 mL to 650 mL, or 650 mL to 700 mL, or 700 mL to750 mL, or 750 mL to 800 mL, or 800 mL to 850 mL, or 850 mL to 900 niL,or 900 mL to 950 mL, or 950 mL to 1000 mL, or 1 mL to 25 mL, or 1 mL to50 mL, or 1 mL to 75 mL, or 1 mL to 100 mL, or 10 mL to 25 mL, or 10 mL50 mL, or 10 mL to 75 mL, or 100 mL to 250 mL, or 100 mL to 500 mL, or100 mL to 750 mL, or 100 mL to 1000 mL.

In some embodiments, the invention provides for administering a tissuefiller disclosed herein. As used herein, the term “administering” meansany delivery mechanism that provides a tissue filler disclosed herein toan individual that potentially results in a clinically, therapeutically,or experimentally beneficial result. The actual delivery mechanism usedto administer a tissue filler to an individual can be determined by aperson of ordinary skill in the art by taking into account factors,including, without limitation, the type of condition, the location ofthe condition, the cause of the condition, the severity of thecondition, the degree of relief desired, the duration of relief desired,the particular tissue filler used, the rate of biodegradability,bioabsorbability, bioresorbability, and the like, of the particulartissue filler used, the nature of the components included in theparticular tissue filler used, the particular route of administration,the particular characteristics, history and risk factors of the patient,such as, e.g., age, weight, general health and the like, or anycombination thereof. In an aspect of this embodiment, a tissue fillerdisclosed herein is administered to a region of a patient by injection,wherein the region may be in the skin, dermal tissues, subdermaltissues, cutaneous tissues, subcutaneous tissues, intradural tissue,muscles, tendons, ligaments, fibrous tissues, fat, blood vessels andarteries, nerves, or synovial (intradermal) tissues.

In some embodiments, the route of administration of a tissue filleradministered to a patient will be determined based on the cosmeticand/or clinical effect desired by the patient and/or physician and thebody part or region being treated. A tissue filler disclosed herein maybe administered by any means known to persons of ordinary skill in theart including, without limitation, syringe with needle, catheter,topically, or by direct surgical implantation. The tissue fillerdisclosed herein can be administered into a skin region such as, e.g., adermal region or a hypodermal region. In addition, a tissue fillerdisclosed herein may be administered once, twice, thrice, or a pluralityof times as required by the specific therapy.

In some embodiments, a tissue filler disclosed herein is injectable. Asused herein, the term “injectable” refers to a tissue material havingthe properties necessary to administer the tissue filler into a skinregion of an individual using an injection device with a needle such as,for example, a fine needle. As used herein, the term “fine needle”refers to a needle that is 27 gauge or smaller. In some embodiments, afine needle can be a 27 gauge to 30 gauge needle. Injectability of atissue filler disclosed herein can be accomplished by varying certainparameters of the tissue filers disclosed herein by, for example,adjusting the degree of cross-linking, otherwise varying G′ and/or G″parameters, adding non-cross linked polymers (e.g., SPF or HA), and thelike.

In some embodiments, a tissue filler disclosed herein is injectablethrough a fine needle. In some embodiments, a tissue filler disclosedherein is injectable through a needle of, for example, 20 gauge, or 21gauge, or 22 gauge, or 23 gauge, or 24 gauge, or 25 gauge, or 26 gauge,or 27 gauge, or 28 gauge, or 29 gauge, or 30 gauge, or 31 gauge, or 32gauge, or 33 gauge, or 34 gauge. In some embodiments, the tissue fillerdescribed herein are injectable through a needle of 20 gauge, or 21gauge, or 22 gauge, or 23 gauge, or 24 gauge, or 25 gauge, or 26 gauge,or 27 gauge, or 28 gauge, or 29 gauge, or 30 gauge.

In some embodiments, a tissue filler disclosed herein is injectable witha syringe having a volume of about 0.8 to about 1.0 mL.

In some embodiments, the tissue fillers described herein may bedelivered to void spaces in or about soft tissues for the purpose of,for example, tissue augmentation (e.g., breast or buttock augmentation).When delivering the tissue fillers described herein to such void spaces,larger syringes and needles may be used (e.g., needles that are 27 gaugeor larger).

In some embodiments, the tissue fillers described herein may be appliedto a wound without the use of a needle in order to coat the wound or amedical device proximate to the wound.

In some embodiments, the tissue fillers described herein may be appliedto a surface of a medical device.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described embodiments, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

EXAMPLES Example 1: Tyndall Evaluation of Gels

In order to further support visual observations and carry outcomparative performance analysis of dermal fillers, quantitativeanalysis of Tyndall effect is performed. Based on existing scientificunderstanding on light scattering and interaction of light with skin,two distinct approaches based on (a) colorimetry, and (b) spectroscopyare employed to quantify Tyndall effect in skin. Based on thesetechniques three distinct quantitative parameters (outlined below) aredefined to measure Tyndall effect in vivo.

Tyndall Effect Visual Score:

The scale has a range of 1 to 5 with increments of 0.5. A score of 1 isgiven to injection sites with normal skin tone and no bluediscoloration. A maximum score of 5 is given to thick and pronouncedblue discoloration. Three independent observers are trained on the scalebefore being blinded to score test samples.

Blue Component of Skin Color—“b”: a chromameter is used to quantify theblue color component of light remitted from skin sites injected with thevarious fillers. This is achieved by using the “b” component of L-a-bcolor scale.

“% Blue Light” Remitted from Skin: a portable spectrophotometer is usedto quantify the % blue light remitted from skin in the total visiblelight range. This is achieved by integrating the area under the visiblelight spectrum between 400-490 nm and normalizing it by the total areaunder the spectrum (400-700 nm).

Gels of the present disclosure and commercially available gels areinjected intradermally through an appropriate needle using linearthreading technique into the thighs of two months old hairless rats. Thegels are implanted superficially to mimic clinical fine line procedures.Tests for Tyndall are performed 48 h after gel implantation. Beforeperforming the Tyndall tests, the animals are humanely euthanized toimprove contrast of the Tyndall effect.

A visual score of 1-5 with increments of 0.5, is used to score theinjection sites. Injection sites with score of 1 show no skindiscoloration, while injections sites with score of 5 show severe bluediscoloration of the skin. Spectroscopic analysis are also performed onthe injection sites with the aid of a chromatometer. The blue componentof skin color “b”, and the % of blue light remitted from skin (400-700nm) are independently measured.

Example 2: In Vivo Tissue Filler Testing

Tissue fillers prepared according to the foregoing description could betested following intradermal implantation, muscle implantation, andsubcutaneous injection.

For example, a dose of a tissue filler could be loaded in a syringe andinjected either intradermally, intramuscularly, or subcutaneously usingan appropriately sized syringe that permits flow through the needle ofthe tissue filler to the injection site.

Following initial injection versus a control (e.g., water and/or amarketed HA based tissue filler such as Juvederm), the injection sitesmay be monitored at 1 week or 2 week intervals where the patients areobserved for biocompatibility concerns, including, cytotoxicity,pyrogenicity, endotoxin formation, acute system toxicity, subchronictoxicity, intradermal reactivity, genotoxicity, and skin sensitization.

In addition, the physical attributes of the tissue filler may bemonitored by examining presence of Tyndalling or loss in volume,elasticity, or firmness at the injection site.

Example 3: Examination of Tissue Filler Rheology

An oscillatory parallel plate rheometer (Anton Paar Physica MCR 301)could be used to measure the rheological properties of the tissuefillers described herein. A plate diameter of 25 mm could be used at agap height of 1 mm. Measurements could be performed at a constanttemperature of 25° C. Each measurement would consist of a frequencysweep from 1 to 10 Hz at a constant strain of 2% and a logarithmicincrease of frequency followed by a strain sweep from 1 to 300% at aconstant frequency of 5 Hz with a logarithmic increase in strain. Theresults of such analyses would provide the Storage Modulus G′ and LossModulus G′ of each tested tissue filler.

Example 4: Examination of Silk/HA Solution Opacity

Solutions of HA and silk were prepared in water or phosphate-bufferedsaline according to Table 18.

TABLE 18 Sample Description 1 Silk MW: “Mid” Silk Conc: 0.3 mg/mL HAConc: 22 mg/mL Solvent: Water 2 Silk MW: “Mid” Silk Conc: 0.6 mg/mL HAConc: 22 mg/mL Solvent: Water 3 Silk MW: “Mid” Silk Conc: 3.0 mg/mL HAConc: 22 mg/mL Solvent: Water 4 Silk MW: “Mid” Silk Conc: 6.0 mg/mL HAConc: 22 mg/mL Solvent: Water 5 Silk MW: “Mid” Silk Conc: 15.0 mg/mL HAConc: 22 mg/mL Solvent: Water 6 Silk MW: “Mid” Silk Conc: 30.0 mg/mL HAConc.: 22 mg/mL Solvent: Water 7 Silk MW: “Mid” Silk Conc: 45.0 mg/mL HAConc: 22 mg/mL Solvent: Water 8 Silk MW: “Low” Silk Conc: 0.6 mg/mL HAConc: 22 mg/mL Solvent: Water 9 Silk MW: “Low” Silk Conc: 15.0 mg/mL HAConc: 22 mg/mL Solvent: Water 10 Silk MW: “Low” Silk Conc: 30.0 mg/mL HAConc: 22 mg/mL Solvent: Water 11 Silk MW: “Low” Silk Conc: 45.0 mg/mL HAConc.: 22 mg/mL Solvent: Water 12 Silk MW: “Mid” Silk Conc: 0.6 mg/mL HAConc: 22 mg/mL Solvent: PBS 13 Silk MW: “Mid” Silk Conc: 15 mg/mL HAConc: 22 mg/mL Solvent: PBS 14 Silk MW: “Mid” Silk Conc: 30.0 mg/mL HAConc: 22 mg/mL Solvent: PBS 15 Silk MW: “Mid” Silk Conc: 45.0 mg/mL HAConc: 22 mg/mL Solvent: PBS Low = silk molecular weights of 0 and 25 kDaMid = silk molecular weights of 25 kDa to 60 kDa

The results the solutions described in the above-table are shown inFIGS. 26 and 27. The control in FIGS. 26 and 27 (unlabeled flask in FIG.26 and control syringe in FIG. 27) was a solution of HA (22 mg/mL) inwater. As illustrated the FIGS. 26 and 27, silk/HA solutions werehomogenous and visibly opaque as compared to HA alone.

Example 5: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Hyaluronic acid may be dissolved in NaOH solution and added to asolution of silk as described herein;

Step b: Add dissolved BDDE in NaOH to Silk/HA/NaOH solution:

Step c: Cross link by mixing with heat;

Step d: Pass through a metal mesh and allow to swell in water;

Step e: Precipitate swelled gel in ethanol;

Step f: Wash with ethanol, water, and NaOH solution;

Step g: Finalize crosslinking in solution of ethanol/NaOH for about 2hours with heating (50° C.);

Step h: Neutralize solution pH to 7;

Step i: Precipitate is washed and dried;

Step j: Resulting dry powder allowed to swell into a gel in buffered0.9% NaCl solution; and

Step k: Gel is filled into a syringe and autoclaved provide resultingtissue filler.

Example 6: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Hyaluronic acid may be dissolved in NaOH solution;

Step b: Add silk in NaOH solution to a solution of Silk, and then adddissolved BDDE in NaOH to Silk/HA/NaOH solution;

Step c: Cross link by mixing with heat:

Step d: Pass through a metal mesh and allow to swell in water;

Step e: Precipitate swelled gel in ethanol;

Step f: Wash with ethanol, water, and NaOH solution:

Step g: Finalize crosslinking in solution of ethanol/NaOH for about 2hours with heating (50° C.);

Step h: Neutralize solution pH to 7;

Step i: Precipitate is washed and dried:

Step j: Resulting dry powder allowed to swell into a gel in buffered0.9% NaCl solution; and

Step k: Gel is filled into a syringe and autoclaved provide resultingtissue filler.

Example 7: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Hyaluronic acid may be dissolved in NaOH solution;

Step b: Add dissolved BDDE in NaOH to HA/NaOH solution;

Step c: Add silk solution to solution of Step b and cross link by mixingwith heat;

Step d: Pass through a metal mesh and allow to swell in water;

Step e: Precipitate swelled gel in ethanol;

Step f: Wash with ethanol, water, and NaOH solution;

Step g: Finalize crosslinking in solution of ethanol/NaOH for about 2hours with heating (50° C.);

Step h: Neutralize solution pH to 7;

Step i: Precipitate is washed and dried;

Step j: Resulting dry powder allowed to swell into a gel in buffered0.9% NaCl solution; and

Step k: Gel is filled into a syringe and autoclaved provide resultingtissue filler.

Example 8: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Hyaluronic acid may be dissolved in NaOH solution;

Step b: Add dissolved BDDE in NaOH to HA/NaOH solution;

Step c: Cross link by mixing with heat;

Step d: Add silk solution to cross-linked HA/NaOH solution, and passthrough a metal mesh and allow to swell in water;

Step e: Precipitate swelled gel in ethanol;

Step f: Wash with ethanol, water, and NaOH solution;

Step g: Finalize crosslinking in solution of ethanol/NaOH for about 2hours with heating (50° C.);

Step h: Neutralize solution pH to 7;

Step i: Precipitate is washed and dried;

Step j: Resulting dry powder allowed to swell into a gel in buffered0.9% NaCl solution; and

Step k: Gel is filled into a syringe and autoclaved provide resultingtissue filler.

Example 9: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Hyaluronic acid may be dissolved in NaOH solution;

Step b: Add dissolved BDDE in NaOH to HA/NaOH solution:

Step c: Cross link by mixing with heat;

Step d: Pass through a metal mesh and allow to swell in water:

Step e: Precipitate swelled gel in ethanol;

Step f: Wash with ethanol, water, and NaOH solution:

Step g: Add silk solution to material prepared in Step f and finalizecrosslinking in solution of ethanol/NaOH for about 2 hours with heating(50° C.);

Step h: Neutralize solution pH to 7;

Step i: Precipitate is washed and dried:

Step j: Resulting dry powder allowed to swell into a gel in buffered0.9% NaCl solution;

Step k: Gel is filled into a syringe and autoclaved provide resultingtissue filler.

Example 10: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Sodium hyaluronate may be mixed with NaOH solution and asolution of silk as described herein;

Step b: BDDE may be added to the solution of Step a;

Step c: The product of Step b is allowed to react;

Step d: Ammonia is added to the dialyzed mixture of Step c and themixture is poured into a petri dish;

Step e: The product of Step d is allowed to dry into a film;

Step f: The film of Step e is divided into particles and swelled insaline;

Step g; The product of Step f is added to a syringe and autoclaved;

Step h (optional): The product of Step f can be subjected to a second,final crosslinking procedure with a solution of BDDE, or othercrosslinking agent described herein, and washed.

Example 11: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Sodium hyaluronate may be mixed with NaOH solution;

Step b: A silk solution may be added to the solution of Step a and BDDEmay be added;

Step c: The product of Step b is allowed to react;

Step d: Ammonia is added to the dialyzed mixture of Step c and themixture is poured into a petri dish:

Step e: The product of Step d is allowed to dry into a film;

Step f: The film of Step e is divided into particles and swelled insaline;

Step g; The product of Step f is added to a syringe and autoclaved;

Step h (optional): The product of Step f can be subjected to a second,final crosslinking procedure with a solution of BDDE, or othercrosslinking agent described herein, and washed.

Example 12: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Sodium hyaluronate may be mixed with NaOH solution;

Step b: BDDE may be added to the solution of Step a;

Step c: The product of Step b is added to a silk solution and allowed toreact;

Step d: Ammonia is added to the dialyzed mixture of Step c and themixture is poured into a petri dish;

Step e: The product of Step d is allowed to dry into a film;

Step f: The film of Step e is divided into particles and swelled insaline;

Step g: The product of Step f is added to a syringe and autoclaved;

Step h (optional): The product of Step f can be subjected to a second,final crosslinking procedure with a solution of BDDE, or othercrosslinking agent described herein, and washed.

Example 13: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: Sodium hyaluronate may be mixed with NaOH solution;

Step b: BDDE may be added to the solution of Step a;

Step c: The product of Step b is allowed to react;

Step d: The product of Step c is added to a silk solution and thenammonia is added to the dialyzed mixture thereof and the mixture ispoured into a petri dish:

Step e: The product of Step d is allowed to dry into a film;

Step f: The film of Step e is divided into particles and swelled insaline;

Step g: The product of Step f is added to a syringe and autoclaved;

Step h (optional): The product of Step f can be subjected to a second,final crosslinking procedure with a solution of BDDE, or othercrosslinking agent described herein, and washed.

Example 14: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: A silk solution may be prepared as described herein, to whichBDDE may be added in water:

Step b: HA may be added to the solution of Step a:

Step c: The mixture of Step b may be stirred (e.g., 5 minutes) andallowed to stand for about 1 day;

Step d: The resulting gel from Step c may be allowed to stand in salinefor 1 week to provide the resulting tissue filler.

Example 15: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: BDDE may be added to water:

Step b: A silk solution may be added to the solution of Step a, to whichHA may then be added;

Step c: The mixture of Step b may be stirred (e.g., 5 minutes) andallowed to stand for about 1 day;

Step d: The resulting gel from Step c may be allowed to stand in salinefor 1 week to provide the resulting tissue filler.

Example 16: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: BDDE may be added to water;

Step b: HA may be added to the solution of Step a;

Step c: A silk solution may be added to the mixture of Step b and theresulting mixture may be stirred (e.g., 5 minutes) and allowed to standfor about 1 day:

Step d: The resulting gel from Step c may be allowed to stand in salinefor 1 week to provide the resulting tissue filler.

Example 17: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: To a silk solution as described herein may be added HA dissolved(mixed for about 12 hours at 400 rpm) in NaOH solution;

Step b: The solution of Step a may be degassed;

Step c: The solution of Step b may be mixed with a crosslinking agentdescribed herein (e.g., BDDE) at 50° C. for about 10-20 minutes;

Step d: The cross-linked gel is mixed with lidocaine HCl;

Step e: Dialysis of the adjusted cross-linked solution may be carriedout for 3 days, then 2 days with PBS, then 1 day with water;

Step f: The filtered resulting product is then lyophilized to obtainsolids;

Step g: The solids are dissolved in PBS and then incubated;

Step h (Optional): free HA may be added to the product of Step g;

Step i: The resulting product of Step g or h may be sterilized by steamautoclaving.

Example 18: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: HA may be dissolved (mixed for about 12 hours at 400 rpm) inNaOH solution;

Step b: A silk solution may be added to the solution of Step a and theresulting mixture may be degassed.

Step c: The solution of Step b may be mixed with a crosslinking agentdescribed herein (e.g., BDDE) at 50° C. for about 10-20 minutes;

Step d: The cross-linked gel is mixed with lidocaine HCl:

Step e: Dialysis of the adjusted cross-linked solution may be carriedout for 3 days, then 2 days with PBS, then 1 day with water;

Step f: The filtered resulting product is then lyophilized to obtainsolids:

Step g: The solids are dissolved in PBS and then incubated:

Step h (Optional): free HA may be added to the product of Step g;

Step i: The resulting product of Step g or h may be sterilized by steamautoclaving.

Example 19: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: HA may be dissolved (mixed for about 12 hours at 400 rpm) inNaOH solution;

Step b: The solution of Step a may be degassed;

Step c: A silk solution may be added to the solution of Step b and theresulting mixture may be mixed with a crosslinking agent describedherein (e.g., BDDE) at 50° C. for about 10-20 minutes;

Step d: The cross-linked gel is mixed with lidocaine HCl;

Step e: Dialysis of the adjusted cross-linked solution may be carriedout for 3 days, then 2 days with PBS, then 1 day with water;

Step f: The filtered resulting product is then lyophilized to obtainsolids;

Step g: The solids are dissolved in PBS and then incubated;

Step h (Optional): free HA may be added to the product of Step g;

Step i: The resulting product of Step g or h may be sterilized by steamautoclaving.

Example 20: Proposed Tissue Filler Preparation Method

A Silk/HA tissue filler as described herein could be prepared accordingto the following general method:

Step a: HA may be dissolved (mixed for about 12 hours at 400 rpm) inNaOH solution;

Step b: The solution of Step a may be degassed;

Step c: The solution of Step b may be mixed with a crosslinking agentdescribed herein (e.g., BDDE) at 50° C. for about 10-20 minutes;

Step d: A silk solution may be added to the product of Step c andmixture may be mixed with lidocaine HCl;

Step e: Dialysis of the adjusted cross-linked solution may be carriedout for 3 days, then 2 days with PBS, then 1 day with water;

Step f: The filtered resulting product is then lyophilized to obtainsolids:

Step g: The solids are dissolved in PBS and then incubated;

Step h (Optional): free HA may be added to the product of Step g;

Step i: The resulting product of Step g or h may be sterilized by steamautoclaving.

Example 21: Dermal Filler Formulations Composed of Silk and HyaluronicAcid Cross Linked with BDDE

Materials: 1,4-butanediol diglycidyl ether (BDDE; Sigma-Aldrich); sodiumhyaluronate (HA: Lifecore); silk, 6% solution (Silk Therapeutics);sodium hydroxide, 0.1 N solution (BDH); hydrochloric acid, 5 N (RiccaChemical); phosphate buffered saline (PBS: 20×; VWR Life Science).

Formulation variables: Silk Molecular Weight: Medium and Low MW silksolution (6%); HA Molecular Weight: 1.5 MDa and 2.2 MDa; Silkconcentration: 1%_(v/v) (0.6 mg/ml), 2%_(v/v) (6 mg/ml) 5%_(v/v) (3mg/ml) and 20%_(v/v) (12 mg/ml).

Hydrogel crosslinking: (a) add 6% silk solution to 0.1 N sodiumhydroxide; (b) gradually add HA powder to above prepared solution underoverhead stir at the speed of 200-400 rpm, depending on the silkcontent; stir gently to avoid generating too much air bubbles; keepstirring until HA is fully dissolved; (c) add 1%_(w/w) of BDDE to theabove solution; (d) heat to 50° C. and keep stirring at 100-200 rpm for30 minutes; (e) let the cross-linked gel cool down below 30° C.; (f) add5N hydrochloric acid to adjust pH to 7.0-7.4.

Hydrogel dialysis: (a)hydrate the dialysis cassette for 2 minutes; wipeoff excessive water and measure the total mass of the empty cassette;(b) add approximately 18 g of hydrogel formulation into the dialysiscassette; measure the total mas of the cassette after is loaded withgel; (c) suspend dialysis cassette in 2 L of 1×PBS buffer and setmagnetic stir at 200 rpm; record the time when dialysis starts andchange the PBS buffer after 4 hrs, 24 hrs, and 48 hrs of dialysis;collect the gel after 72 hrs.

Characterization: shear storage modulus (G′) and viscosity; enzymaticdegradation; BDDE residual; crosslinking density; 30-day animal study;cytotoxicity; bacterial endotoxin; turbidity.

Viscoelastic properties: A Discovery HR-1 hybrid rheometer (TAInstruments) was used to determine storage modulus (G′) and complexviscosity (η) of dermal filler formulations. Samples were tested byswiping oscillation frequency from 0.1 Hz to 10 Hz with 10 data pointsper decade interval. Data were recorded and compared at 5 Hz shear rate.The G′ and γ data for hydrogel formulations (after dialysis) withconstant HA concentration and variable silk concentration are shown inTable 19. In this batch, 1.5 MDa molecular weight HA was used.

TABLE 19 Viscoelastic properties of hydrogels with constant HAconcentration HA Conc.* Silk. Conc.* G′ at 5 Hz η at 5 Hz Sample (mg/ml)(mg/ml) Silk MW (Pa) (Pa · s) C2 24 0 N/A 46.9 2.88 A 24 9.6 Medium105.5 4.93 B 24 0.48 Low 69.7 3.62 C 24 4.8 Medium 102.7 4.82 D 24 0.48Medium 66.4 3.59 E 24 2.4 Low 41.4 9.56 F 24 0.96 Low 42.7 2.67*Hydrogel absorbed PBS buffer after dialysis resulting in volumeincrease. The concentration of HA and silk were recalculated based onthe dilution factor.

The G′ and γ data for hydrogel formulations (after dialysis) withconstant total concentration of 30 mg/ml of HA and silk are summarizedin Table 20.

TABLE 20 Viscoelastic properties of hydrogels with constant totalconcentration HA HA Silk G′ at η at Conc.* MW Conc.* Silk % 5 Hz 5 HzSample (mg/ml) (MDa) (mg/ml) MW Silk (Pa) (Pa · s) XHA15M01SL1712200223.52 1.5 0.48 Low  1% 94.1 4.52 XHA15M05SL17112002 21.60 1.5 2.4 Low 5% 29.5 2.06 XHA15M20SL17122002 14.40 1.5 9.6 Low 20% 31.7 1.63XHA15M01SM17121802 23.52 1.5 0.48 Medium  1% 118.1 5.55XHA15M05SL17111602 21.60 1.5 2.4 Medium  5% 38.4 2.35 XHA15M20SM1711270214.40 1.5 9.6 Medium 20% 15.6 1.06 XHA2M01SL171121902 23.52 2.2 0.48 Low 1% 176.3 7.50 XHA2M05SL17122002 21.60 2.2 2.4 Low  5% 85.1 4.03XHA2M20SM17122002 14.40 2.2 9.6 Low 20% 36.0 1.76 XHA2M01SM1712190223.52 2.2 0.48 Medium  1% 158.1 6.69 XHA2M05SM17122002 21.60 2.2 2.4Medium  5% 106.7 4.76 XHA2M20SM17111302 14.40 2.2 9.6 Medium 20% 11.50.86 *Hydrogel absorbed PBS buffer after dialysis resulting in volumeincrease. The concentration of HA and silk were recalculated based onthe dilution factor.

Hydrogel reversibility: Hydrogels with and without silk protein wereprepared and dialyzed. The final compositions were 33.3 mg/ml HA+8 mg/mlsilk for Silk-HA hydrogel and 33.3 mg/ml of HA for HA hydrogel,respectively. 1 g±100 g of above prepared hydrogels were added to 20 mlglass vial followed by 3 ml of 16 U/ml of hyaluronidase in 1×PBS.Samples were incubated at 37° C. for 3 days. Control samples was alsoprepared using HA hydrogel without adding hyaluronidase. The degradationprofile is shown in FIG. 28. Control samples without hyaluronidase wasnot degraded during the course of 3 days incubation. Within the first 6hours of incubation, hydrogels absorbed buffer and swelled resulting inthe increase of percentage mass. The Silk-HA hydrogel and HA hydrogelwere fully degraded after 3 days incubation. At the presence of silk,the hydrogel was digested faster than the pure HA hydrogel. After 12hours of incubation, approximately 90% of the Silk-HA hydrogel wasdigested by enzyme.

Crosslinker (BDDE) residual: Samples listed in Table 19 were tested forBDDE residuals using GC-FID by Millennium Research Laboratories, Inc.(MRL). MRL test report MRL18JAN06 indicated that BDDE residual in allsamples were none detectable, meeting the acceptance criteria of equalto or less than 2 ppm.

Crosslinking density: Samples listed in Table 19 were further fullydigested by hyaluronidase and analyzed using NMR to determine thecrosslinking density in term of percentage modification. The testresults are listed in Table 21 (MRL test report MRL18JAN07).

TABLE 21 Percentage modification degree (crosslinking density) forvarious formulations Sample ID MoD (%) XEIA15M00SX17110202 (C2) 2.87XFIA15M205M17103002 (A) 4.68 XHA15M01SL17103002 (B) 2.58XFIA15M10SM17103002 (C) 3.02 XEIA15M01SM17103002 (D) 7.54XFIA15M05SL17110202 (E) 3.76

Animal study: A 30-day animal study using guinea pig model was carriedout at WuXi AppTec Minneapolis, Minn. facility to address product safetyconcern. There were 2 termination time points in this study, 7 days and30 days, to evaluate tissue response. The study was summarized in WuXiAppTec report D28195 (Project C19879). Two control samples (JuvedermUltra Plus and Sample C2 in Table 19) and 6 formulations (Sample A-F inTable 19) were used for intradermal injection. Samples A-F and controlsample C2 were steam sterilized (protocol 201707289) at NelsonLaboratories, LLC prior to injection. The study procedure in brief:twenty-four animals twelve per duration were used in this study. Eachanimal received six dorsal, intradermal injections using threadingtechnique (injecting a line instead of a bolus): one control site on oneside of the spine, the second control site on the contralateral side(with sides alternatively assigned by animal) and four test sites withno more than one injection of a given test article (with right and leftsides alternatively assigned among animals). Animals were observed dailythroughout the study to assess general health. Animals were humanelyeuthanized at the scheduled termination dates. The implant sites andsurrounding tissue from all animals were excised, placed in formalin,and processed to paraffin blocks followed by histopathologicalevaluation. The representative histology images and pathologicalfindings were summarized in Table 22. Overall, there was no suggestionof sepsis or immunological response in any of the implant sites.

TABLE 22 Summary of histopathological evaluations Samples 7 Days 30 DaysCommercial FIG. 29 FIG. 30 Control The Commercial product is noted TheCommercial product is in both images as blue/gray noted in both imagesas blue/gray material. There is mild material. At 30 days, there is agranulomatous inflammation minimal amount of inflammation associatedwith the material at 7 with very mild fibrosis. days. Product A: FIG. 31FIG. 32 24 mg/ml HA There is very little inflammation At 30 days theinflammation is 9.6 inglinl silk at 7 days. The inflammation wasextremely difficult to find and focal and at times hard to find.minimal. No implant material is No implant material is noted. noted.Product B: FIG. 33 FIG. 34 24 mg/ml HA Product B demonstrates focal The30-day image demonstrates 0.48 mg/ml silk mild inflammation in the 7days. even less inflammation. It was The inflammation is chronic, evenmore difficult to identify as This inflammation required close comparedto the 7 day implants. evaluation to identify since it was No implantmaterial is observed. focal and minimal. No implant material isobserved. Overall, there was no suggestion of sepsis or innnunohogicresponse in any of the implant sites.

Bacterial endotoxin: Three post sterilization samples (Sample A, SampleE and Sample C2) were selected from 7 formulations used in animal study(listed in Table 19) for bacterial endotoxin test. The kineticTurbidimetric method was used to determine endotoxin level. Test resultsare listed in Table 23, and are below the acceptance criteria of 20EU/ml (Nelson Labs study report 1006775-S01).

TABLE 23 Endotoxin test results Sample ID Detected EndotoxinXHA15M20SM17103002 (A)   0.498 (EU/ml) XHA15M00SX17110202 (C2) <0.400(EU/ml) XHA15M05SL17110202 (E)    1.56 (EU/ml)

Biocompatibility—Cytotoxicity: Four post sterilization samples (SampleA, Sample B, Sample D and Sample E) were selected from 7 formulationsused in animal study (listed in Table 19) for ISO-10993-5 cytotoxicitytest (ISO MEM Elution Using L-929 Mouse Fibroblast Cells). These samplesrepresented the highest and lowest silk content of medium molecularweight silk and low molecular weight silk in tested dermal fillerformulations. The test reports indicated that all test samples scoredgrade 0, meaning non-cytotoxic (Wuxi AppTec Reports D28287-1, D28287-2.D28287-3, D28287-4).

Turbidity: The pure HA hydrogel is clear under natural light. When HAwas cross-linked with silk protein, the hydrogel becomes slightly turbid(cloudy) and the turbidity is dependent on the total silk concentrationin the formulation. The turbidity was measured by Lambda X50S UV-Visspectrophotometer (PerkinElmer) equipped with InGaAs integrating spherewhich has the capability to collect forward scattered light in additionto standard transmitted light. The turbidity measurement of Silk-HAhydrogel is shown in FIG. 35. The black curve is the standardtransmittance and the red curve was collected by the sphere showingsignificant forward scatter. The pure HA hydrogel without silk was usedas control sample. The curves in FIG. 36 are nearly identical indicatingvery little scattering of the pure HA gel. The turbidity measurementsuggested that the Silk-HA hydrogel has the capability of scatteringlights which could eliminate Tyndall effect when used as dermal filler.

Conclusions: Dermal filler formulations were developed based on constantHA concentration with various silk contents and constant totalconcentration. These formulations provided a broad range of storagemodulus, viscosity and crosslinking density which may lead to variousapplications. The silk-HA hydrogel was enzymatically reversible. Thecrosslinker residual after dialysis of hydrogel formulations met theacceptance criteria. Cytotoxicity test indicated that silk-HA hydrogelswith of silk content ranging from 0.48 mg/ml to 9.6 mg/ml were nonecytotoxic and biocompatible. The 30-day animal study demonstrated allformulations with silk content up to 9.6 mg/ml did not cause sepsis andhad no immunological response.

Example 22: Dermal Filler Formulations Composed of Silk and HyaluronicAcid Cross Linked with PEGDE (PEGDGE)

Crosslinker: Poly(ethylene glycol) diglycidyl ether (PEGDE), averagemolecular weight Mn=500. Reaction conditions: same as BDDE crosslinking(Example 21). The total amount of PEGDE was equivalent to BDDE in moles.

TABLE 24 PEGDE cross linking formulation and test results HA HA Silk G′at η at Conc.* MW Conc.* Silk % Cross- 5 Hz 5 Hz Sample (mg/ml) (MDa)(mg/ml) MW Silk linker (Pa) (Pa · s) XHA15M05SM17111602 21.60 1.5 2.4Medium 10% BDDE 38.4 2.35 XHA15M05SM18020802P 20.45 1.5 2.27 Medium 10%PEG-x 67.5 3.10 XHA15M05SM18020902P 19.28 1.5 2.14 Medium 10% PEG-x 73.53.40 *Hydrogel absorbed PBS buffer after dialysis resulting in volumeincrease. The concentration of HA and silk were recalculated based onthe dilution factor.

Example 23: Animal Study C20419

Formulations and characterization of samples for animal study C20419 areas shown in Table 25:

TABLE 25 Formulations and characterization of samples for animal studyC20419 Injection Force HA Silk HA G′ at η at @ Cross- Conc.* Conc.* MWSilk 5 Hz 5 Hz 30 G MoD Sample ID linker (mg/ml) (mg/ml) (Da) MW (Pa)(Pa · s) (N) (%) Group C3 XHA700K3M00SX180510 BDDE 24 0 700K/3M n/a 0.20.1 7.41 4.87 1 G XHA700K3M05SX180510 22.8 1.2 700K/3M Med 3.8 0.2 8.174.54 H XHA700K3M01SL180510 23.76 0.24 700K/3M Low 0 0.1 6.95 5.42 IXHA700K3M05SL180510 22.8 1.2 700K/3M Low 0.5 0.1 7.96 6.23 KXHA26M05SM180510 22.8 1.2 2.6M Med 0.1 0.1 8.48 2.51 Group C4PXHA700K3M00SX180514 PEGDE 24 0 700K/3M n/a 52.3 2.4 16.19 15.14 2 LPXHA700K3M05SX180514 22.8 1.2 700K/3M Med 31.8 1.6 12.96 10.97 MPXHA700K3M01SL180514 23.76 0.24 700K/3M Low 32.2 1.5 15.96 11.02 NPXHA700K3M05SL180514 22.8 1.2 700K/3M Low 51.9 2.1 17.82 11.23 OPXHA26M05SM180514 22.8 1.2 2.6M Med 18.9 1.1 10.56 17.23 Group C5 Group2 + Free HA 700K/3M n/a 63.0 2.8 19.02 8.02 3 P 700K/3M Med 28.3 1.411.22 9.71 Q 700K/3M Low 42.7 1.9 16.80 10 R 700K/3M Low 83.9 3.2 20.9010.12 S 2.6M Med 75.8 3.4 12.78 11.92

FIGS. 37-46 show the results of the study. FIG. 37 is a representativehistology picture of an intradermal area in a guinea pig injected with acontrol dermal filler. FIG. 38 is a representative histology picture ofan intradermal area in a guinea pig injected with an HA dermal filler ofthe invention (24 mg/ml HA, PEGDE cross linked. Sample C4—Table 25).FIG. 39 is a representative histology picture of an intradermal area ina guinea pig injected with a silk-HA dermal filler of the invention(22.8 mg/ml HA, 1.2 mg/ml silk, PEGDE cross linked, Sample L—Table 25).FIG. 40 is a representative histology picture of an intradermal area ina guinea pig injected with a silk-HA dermal filler of the invention(23.76 mg/ml HA, 0.24 mg/ml silk, PEGDE cross linked, Sample M—Table25). FIG. 41 is a representative histology picture of an intradermalarea in a guinea pig injected with a silk-HA dermal filler of theinvention (22.8 mg/ml HA, 1.2 mg/ml silk, PEGDE cross linked, SampleN—Table 25). FIG. 42 is a representative histology picture of anintradermal area in a guinea pig injected with a silk-HA dermal fillerof the invention (22.8 mg/ml HA, 1.2 mg/ml silk, PEGDE cross linked,Sample O—Table 25).

FIGS. 43-46 are graphical representations of histology results for Table25 formulations 7-day post-implantation (scoring: 0—normal: 1—minimal;2—mild; 3—moderate; and 4-severe). FIG. 43 is a graph showing 7-daypost-implantation histology results for gel degradation; BDDEcross-linked formulations are mostly degraded. FIG. 44 is a graphshowing 7-day post-implantation histology results for gel migration.FIG. 45 is a graph showing 7-day post-implantation histology results forinflammation; no tissue necrosis was observed, no blood clotting wasobserved, and minimal collagen deposition was observed on the controlformulation and some of the test formulations. FIG. 46 is a graphshowing 7-day post-implantation histology results for macrophages.

Example 24: Properties of PEGDE Cross-Linked Silk-HA Hydrogels: 1) ShearStorage Modulus (G′), and 2) Swelling Ratio During Dialysis

Dermal Filler Preparation, Materials: Poly(ethylene glycol) diglycidylether (PEGDE), Mn=500, Sigma-Aldrich; Sodium hyaluronate (HA), Lifecore;Silk, 6% solution, Silk Inc.; Sodium hydroxide, 0.1 N solution, BDH;Hydrochloric acid, 5 N, Ricca Chemical; Phosphate Buffered Saline (PBS),20×, VWR Life Science.

Dermal Filler Formulation variables: Silk Molecular Weight: Medium andLow MW silk solution (6%); HA Molecular Weight: 700 KDa and 1.5 MDa;Silk concentration (Initial): 0-15 mg/ml.

Hydrogel crosslinking at high concentration: add 6% silk solution to 0.1N sodium hydroxide; gradually add 100 mg/ml of mixed molecular weight HA(700 KDa/1.51 MDa=90/10) to the above prepared solution under gentlestirring until HA is fully dissolved, add PEGDE to the above solution;heat water bath to 40° C. and maintain the crosslinking in water bathfor 45 minutes; let the cross-linked gel cool down below 30° C.; add 5Nhydrochloric acid to 1×PBS, dilute the gel to 40 mg/ml and adjust thefinal pH to 7.0-7.4.

Hydrogel crosslinking at low concentration: add 6% silk solution to 0.1N sodium hydroxide; gradually add 25 mg/ml of 1.5 MDa HA to aboveprepared solution under gentle stirring until HA is fully dissolved; addPEGDE to the above solution; heat water bath to 40° C. and maintain thecrosslinking in water bath for 45 minutes; let the cross-linked gel cooldown below 30° C.; add 5N hydrochloric acid to the cross-linked gel andadjust the final pH to 7.0-7.4.

Hydrogel dialysis: hydrate the dialysis cassette (20 KDa MWCO) for 2minutes; wipe off excessive water and measure the total mass of theempty cassette; add approximately 18 g of hydrogel into dialysiscassette; measure the total mass of the cassette after loaded with gel;suspend dialysis cassette in 2 L of 1×PBS buffer and set magnetic stirat 200 rpm; collect gel after 72 hrs of dialysis.

Viscoelastic Properties

A Discovery HR-1 hybrid rheometer (TA Instruments) was used to determinethe storage modulus (G′) of the hydrogel formulations. Samples weretested by swiping oscillation frequency from 0.1 Hz to 10 Hz with 10data points per decade interval. Data were recorded and compared at 5 Hzshear rate. The G′ of hydrogel formulations before and after dialysiswith constant HA concentration and variable silk concentration are shownin FIGS. 47A and 47B. For the hydrogel cross-linked by PEGDE at highinitial HA concentration, the impact of silk concentration to the G′ isminimal due to the relatively low ratio of silk to total HA. It may alsobe contributed to the mixed HA containing 90% of low molecular weight(700 KDa) which is not sensitive to the changes of silk concentration.For the hydrogel cross-linked by PEGDE at low initial HA concentration,the G′ increased as more silk was added to the formulation. The changesin silk concentration had more impact to G′ when the initial HAconcentration was low and also had more impact to the high molecularweight HA (1.5 MDa). No substantial impact of silk molecular weight tothe G′ was observed for both crosslinking procedures.

Swelling ratio during dialysis: there was no clear trend showing theamount of silk added to the hydrogel formulation had any impact to thegel swelling during dialysis for both cross-linking procedures and nosubstantial difference between medium molecular weight and low molecularweight silk (FIGS. 48A and 48B).

The silk concentration in hydrogel formulations had minimal impact to G′if mixed HA was cross-linked by PEGDE at high initial HA concentration,but was proportional to G′ if single high MW HA was cross-linked at lowinitial HA concentration. The molecular weight of silk in the gelformulations had no substantial difference when comparing thecontribution to G′ and swelling if the HA was cross-linked by PEGDE.

Example 25: Silk Concentration in Silk-HA Dermal Filler Formulations

Materials: silk, 6% solution, Silk, Inc.; phosphate buffered saline(PBS), 20×, VWR Life Science; cross-linked hyaluronic acid (HA) gel.

Equipment: moisture analyzer HE53, Mettler Toledo; Cary 100 UV/VisSpectrophotometer.

Calibration Standard Curve: measure the dry content for both medium andlow molecular weight 6% silk solutions to determine the actual drycontent (mg/ml) of the silk solutions; create a series of standard silksolutions by diluting the 6% silk solution using 1×PBS (for example, 1mg/ml silk, 0.75 mg/ml silk, 0.5 mg/ml silk, 0.25 mg/ml silk, and 0mg/ml silk); measure the absorbance of each standard solution at 275 nmin a quartz cuvette—absorbance measurements can be performed with a scanfrom 200-800 nm, data interval of 5 nm, and an average collection of 0.1seconds; Plot the absorbance at 275 nm against the silk concentration(mg/ml) to create a standard curve.

Measurement of Silk Concentration: dilute HA gel samples with 1×PBS suchthat absorbance at 275 nm is between 0 and 1.0 (for example, the samplescan be diluted with a 1:12 ratio of gel to 1×PBS, i.e., 1200% dilution);perform a scan for absorbance for the silk-HA gel sample against a 1×PBSreference between 200 nm-800 nm, measure the absorbance peak at 275 nmfor each gel sample; the absorption signals for the gel samples arecorrected by the difference between the absorption signal for the samplewith no silk and the intercept of the calibration curve, setting thesample with no silk to have a silk concentration of 0 mg/ml; the silkconcentration in the silk-HA gel samples can be calculated from thecalibration curve and dilution factor.

Calibration curves were created by measuring the absorption at 275 nmfor a series of standard samples with different concentrations of silkranging from 0 mg/ml to 1 mg/ml. The calibration curves for the mediumand low molecular weight silk solutions are shown in FIGS. 49A and 49B.The R² values of 0.99947 for medium molecular weight silk and 0.99949for low molecular weight silk demonstrate that the calibration curvesare linear within the working range of 0-1 mg/ml of silk concentration.These curves can be used to determine the silk concentrations in gelsamples.

Determining Silk Concentration of HA-Silk hydrogels: the absorption at275 nm of diluted silk-HA hydrogels was measured for each sample asshown in FIGS. 50A and 50B. The silk concentration of each sample wascalculated with the calibration curve and dilution factor, summarized inTable 26.

TABLE 26 Calculated silk concentrations for silk-HA gels with an unknownsilk concentration from the calibration curve Theoretical SilkCalculated Silk Concentration Concentration Gel Sample (mg/ml) (mg/ml)XTIA15M00SX17110201 0 0 XHA1511401SL17103001 0.6 0.49 XHA15M02SL171102011.2 1.26 XHA15M05SL17110201 3 3.08 XHA15N401SM17103001 0.6 0.57XHA15M10SM17103001 6 6.21 XHA15M20SM17103001 12 13.83

Example 26: Silk-HA Dermal Filler Formulations: Gel Opacity

Materials: cross-linked hyaluronic acid (HA) gel; phosphate bufferedsaline (PBS), 20×. VWR Life Science.

Equipment: Cary 100 UV/Vis Spectrophotometer.

Sample Preparation: inject about 2 mL of HA gel into a clean quartzcuvette such that there is a minimal amount of air bubbles in thesample; injection using an 18 G needle may help reduce the amount ofbubbles in the sample: a blank reference sample of 1×PBS can be added toa second clean quartz cuvette (Note: for opacity measurements, a plasticcuvette can be used since the plastic cuvette does not have absorptionin the visible range, 400 nm-800 nm).

Measurement of Gel Opacity: set the X-scanning range from 200 nm to 800nm with a data interval of 5 nm and average time of 0.1 seconds; selectthe Y-mode to be % T for the measurement of transmitted light (Note:Absorption can also be measured and % T can be calculated fromAbsorption values); perform a scan on the gel sample against the 1×PBSreference standard; the data can be saved as a csv file and the spectrumcan be plotted.

Gel Opacity can be measured using the UV/Vis spectrophotometer forstandard transmitted light. An optically clear sample will transmit100%/o of light, whereas a slightly turbid or cloudy sample may onlytransmit a portion of that light. FIG. 51 shows the turbiditymeasurement of an HA hydrogel with and without silk. The blue curveshows the % transmittance for the transmitted light for a Silk-HA gelsample with 3 mg/mL silk and 26 mg/ml HA. The red curve shows thetransmitted light for a sample with no silk and 20 mg/ml HA, and showsmore transmission of light than the sample with silk. The turbiditymeasurements suggest that the Silk-HA gel has an ability to scattervisible light more than the HA gel without silk.

Example 27: Degree of Modification (MoD) of the HA Hydrogel Determinedby NMR

Degree of Modification (MoD) is defined as the stoichiometric ratio ofall linked cross-linker molecules to the moles of HA repeating units.Both cross- and mono-linked linkers are included in MoD. MoD isdetermined from ¹H NMR spectrum by integrating the signal from theN-acetyl group in HA at 2.1 ppm and the BDDE cross-linker at 1.7 ppm, orthe PEGDE cross-linker at 3.0-4.5 ppm.

Prior to enzymatic degradation, the HA hydrogel was first dialyzed againPBS (1×, 2 L×5) solution to remove the free cross-linker. ASlide-A-Lyzer dialysis cassette (MWCO 3.5 K, Thermo Scientific,Rockford, Ill.) was used, and the PBS solution was stirred at RT for 72h. After the dialysis, 1 mL of the HA hydrogel solution was taken outand lyophilized with a Labconco FreeZone lyophilizer (2.5 L) to obtainthe dry powder.

To prepare the NMR sample, 10 mg of the dry powder was placed into theNMR tube (5 mm, Wilmad-LabGlass) and 0.6 mL of hyaluronidase (MPBiomedicals. Solon, Ohio) solution in deuterium oxide (D₂O, Alfa Aesar,Ward Hill, Mass.) was added. The amount of the hyaluronidase was 5 U per1 mg of HA. The NMR tube was incubated at 37° C. overnight to make allthe HA degraded. The NMR spectra were recorded on a Varian MR 400 MHzAutomated NMR System. The relaxation delay time is 1 s and the number ofscans is 256. All the data was processed using a MestReNova software(Edition 12.0.2).

Example 28: Silk-HA 2-Step Cross-Linking Process

A silk-HA hydrogel can be formed a 2-step crosslinking process toimprove the efficiency of silk binding to HA. For a given formulation,at the first step, all silk protein and a small portion of low molecularweight HA are added to NaOH solution at pH 10, and then reacted with aportion of crosslinker. Without wishing to be bound by any particulartheory, it is believed that during this step, as much silk as possiblereacts with the crosslinker. At the second step, NaOH solution is addedto dilute the product from step-1 and increase the pH to 13. Theremaining low molecular weight HA, all high molecular weight HA, and theremaining crosslinker are then added to the solution, and thecrosslinking reaction is completed.

Example 29: HA Hydrogel Synthesis

HA hydrogel has been synthesized by using different HA molecular weight,crosslinker, reaction time, reaction temperature, HA concentration,crosslinker ratio, mixing process and stirring method. Tables 27 and 28show the various reaction conditions employed, and the various hydrogelsobtained.

TABLE 27 HA MW 700k, 1.5M. 2.2M, 3M, or mixture with different MW at anyratio Crosslinker PEG500DE, and BDDE Reaction time 30 min, 60 min, 90min, 120 min, or 240 min Reaction 40° C., or 50° C. Temperature HAconcentration 30 mg/ml, 90 mg/ml 100 mg/ml, and 140 mg/ml CrosslinkerRatio 7 Wt. % or 10 Wt. % Mixing process Pre-mix HA and crosslinkertogether or adding crosslinker into the HA solution portion wiseStirring With or without mechanical stirring

TABLE 28 Cross- HA/ linker HA Reaction G′ Cross- ratio Concentrationtime Temp (After MoD Sample linker (Wt. %) (mg/mL) Mixing Stirring (min)° C. Dialysis) (%) PXHA2M00SX18042541  2.2M/ 10 30 Portion Y 30 40 16313.02 PEG500DE wise PXHA2M00SX18042543  2.2M/ 10 30 Portion Y 60 40 1069.55 PEG500DE wise PXHA2M00SX18042545  2.2M/ 10 30 Portion Y 120 40 9511.73 PEG500DE wise PXHA2M00SX18042547  2.2M/ 10 30 One Y 240 40 10.615.6 PEG500DE pot PXHA2M00SX18051041  2.2M/ 10 30 One N 30 40 148.67 5.3PEG500DE pot PXHA2M00SX18051043  2.2M/ 10 30 One N 60 40 134.61 7.88PEG500DE pot PXHA2M00SX18051045  2.2M/ 10 30 One N 120 40 46.53 9.44PEG500DE pot PXHA2M00SX18051047  2.2M/ 10 30 One N 240 40 28.9 11.2PEG500DE pot BXHA700K00SX18050141  700K/ 10 30 Drop- Y 30 40 42 0 BDDEwise BXHA700K00SX18050143  700K/ 10 30 Drop- Y 60 40 38 0 BDDE wiseBXHA700K00SX18050145  700K/ 10 30 Drop- Y 120 40 15 0.54 BDDE wisePXHA2M00SX18051641  2.2M/ 10 30 One pot/ N 30 40 182.91 4.62 PEG500DEovernight PXHA2M00SX18051643  2.2M/ 10 30 One pot/ N 60 40 129.76 8.87PEG500DE overnight BXHA700K00SX18052941  700K/ 10 30 Portion N 30 4017.99 0 BDDE wise BXHA700K00SX18052943  700K/ 10 30 Portion N 60 4033.76 0.48 BDDE wise BXHA2M00SX18052941  2.2M/ 10 30 Portion N 30 40295.6 0.52 BDDE wise BXHA2M00SX18052943  2.2M/ 10 30 Portion N 60 40222.28 0.66 BDDE wise BXHA15M00SX18060851  1.5M/ 10 90 Mixed N 30 50261.86 3.26 BDDE separately BXHA15M00SX18060853  1.5M/ 10 90 Mixed N 6050 196.8 3.4 BDDE separately BXHA15M00SX18060855  1.5M/ 10 90 Mixed N 9050 93.6 4.84 BDDE separately BXHA15M00SX18060857  1.5M/ 10 90 Mixed N120 50 72.98 4.51 BDDE separately PXHA15M00SX18061351  1.5M/ 10 90 MixedN 30 50 151.65 under- PEGDE separately going PXHA15M00SX18061351  1.5M/10 90 Mixed N 60 50 71.87 under- PEGDE separately goingBXHA15M00SX18061551  1.5M/ 10 100 One N 30 50 234.69 4.6 BDDE potBXHA15M00SX18061553  1.5M/ 10 100 One N 60 50 219.43 6.1 BDDE potBXHA3M00SX18061951   3M/ 10 100 One N 60 50 268.41 under- BDDE pot goingBXHA3M00SX18061953   3M/ 7 100 One N 60 50 189.13 under- BDDE pot going

All patents, patent applications, and published references cited hereinare hereby incorporated by reference in their entirety. While themethods of the present disclosure have been described in connection withthe specific embodiments thereof, it will be understood that it iscapable of further modification. Further, this application is intendedto cover any variations, uses, or adaptations of the methods of thepresent disclosure, including such departures from the presentdisclosure as come within known or customary practice in the art towhich the methods of the present disclosure pertain.

1-33. (canceled)
 34. A biocompatible tissue filler, comprisinghyaluronic acid (HA), an anesthetic agent, and silk fibroin proteinfragments (SPF), wherein a portion of the HA or SPF is modified orcrosslinked by one or more linker moieties comprising one or more of analkane or alkyl chain, an ether group, and a secondary alcohol, whereinthe linker moieties are covalently attached to the modified orcrosslinked HA or SPF. 35-36. (canceled)
 37. The tissue filler of claim34, wherein the SPF have an average weight average molecular weightranging from about 1 kDa to about 250 kDa.
 38. The tissue filler ofclaim 34, wherein the SPF have an average weight average molecularweight ranging from about 5 kDa to about 150 kDa.
 39. The tissue fillerof claim 34, wherein the SPF have an average weight average molecularweight selected from about 6 kDa to about 17 kDa, from about 17 kDa toabout 39 kDa, and from about 39 kDa to about 80 kDa. 40-44. (canceled)45. The tissue filler of claim 34, wherein the silk fibroin proteinfragments (SPF) have a polydispersity of between about 1.5 and about3.0. 46-50. (canceled)
 51. The tissue filler of claim 34, wherein thedegree of modification or cross-linking of the modified or cross-linkedHA or SPF is between about 1% and about 15%. 52-53. (canceled)
 54. Thetissue filler of claim 34, wherein the modified or cross-linked HA orSPF comprises a linker or cross-linking moiety comprising a polyethyleneglycol (PEG) chain.
 55. The tissue filler of claim 34, wherein themodified or cross-linked HA or SPF comprises a linker or cross-linkingmoiety comprising a secondary alcohol. 56-65. (canceled)
 66. The tissuefiller of claim 34, wherein the tissue filler is a gel.
 67. The tissuefiller of claim 34, wherein the tissue filler is a hydrogel. 68.(canceled)
 69. The tissue filler of claim 34, wherein the totalconcentration of SPF in the tissue filler is from about 0.1 mg/mL toabout 15 mg/mL. 70-74. (canceled)
 75. The tissue filler of claim 34,wherein the tissue filler is injectable.
 76. The tissue filler of claim34, wherein the tissue filler has a storage modulus (G′) of from about25 Pa to about 1500 Pa.
 77. The tissue filler of claim 34, wherein thetissue filler has a storage modulus (G′) of from about 50 Pa to about100 Pa.
 78. The tissue filler of claim 34, wherein the tissue filler hasa storage modulus (G′) of from about 100 Pa to about 200 Pa.
 79. Thetissue filler of claim 34, wherein the tissue filler has a storagemodulus (G′) of from about 200 Pa to about 300 Pa. 80-81. (canceled) 82.The tissue filler of claim 34, wherein the tissue filler has a complexviscosity from about 1 Pa·s to about 10 Pa·s. 83-86. (canceled)
 87. Amethod of treating a condition in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of thetissue filler of claim
 34. 88. (canceled)
 89. The method of claim 87,wherein the condition is a skin condition selected from the groupconsisting of skin dehydration, lack of skin elasticity, skin roughness,lack of skin tautness, a skin stretch line, a skin stretch mark, skinpaleness, a dermal divot, a sunken cheek, a thin lip, a retro-orbitaldefect, a facial fold, and a wrinkle, wherein the tissue filler isadministered into a dermal region of the subject. 90-96. (canceled)